Partial esters of hyaluronic acid

ABSTRACT

The invention concerns the esters of hyaluronic acid in which all or only a portion of the carboxylic groups of the acid are esterified, and the salts of the partial esters with metals or with pharmacologically acceptable organic bases. The compounds possess interesting and precious bioplastic and pharmaceutical properties and may be used in innumerable fields, including cosmetics, surgery and medicine. The invention also includes pharmaceutical preparations containing, as an active ingredient, one or more hyaluronic acid esters, or a salt thereof as described above, as well as medicaments containing: 
     1) a pharmacologically active substance or an association of pharmacologically active substances and 
     2) a carrying vehicle containing a total or partial ester of hyaluronic acid. The invention includes also various uses of the hyaluronic esters or of the above mentioned medicaments, such as in medicine, surgery or cosmetics. 
     The invention also relates to a new procedure for the preparation of polysaccharide esters containing carboxylic groups, such as in particular the above mentioned hyaluronic acid esters.

This application is a continuation of application Ser. No. 07/663,324filed on Mar. 1, 1991, now abandoned, which is a divisional of copendingapplication Ser. No. 07/562,267 filed Aug. 3, 1990, now abandoned, whichis a divisional of application Ser. No. 07/339,919 filed Apr. 19, 1989,now U.S. Pat. No. 4,965,353, which is a divisional of application Ser.No. 06/881,454 filed Jul. 2, 1986, now U.S. Pat. No. 4,851,521. Theentire contents of which are hereby incorporated by reference.

BACKGROUND AND FIELD OF THE INVENTION

The present invention relates to new polysaccharide esters and moreprecisely esters of hyaluronic acid and their use in the pharmaceuticaland cosmetic fields, and in the field of biodegradable plasticmaterials. The invention therefore includes new medicaments, cosmetic,medical and surgical articles.

The term "hyaluronic acid" (also referred to as "HY" hereinafter) isused in literature to designate an acidic polysaccharide with variousmolecular weights constituted by residues of D-glucuronic acid andN-acetyl-D-glucosamine, which naturally occur in cellular surfaces, inthe basic extracellular substances of the connective tissues ofvertebrates, in the synovial fluid of joints, in the vitreous humor ofthe eye, in the tissue of the human umbilical cord and in cocks' combs.

Hyaluronic acid plays an important role in the biological organism,firstly as a mechanical support of the cells of many tissues, such asthe skin, the tendons, the muscles and cartilage and it is therefore themain component of the intracellular matrix. But hyaluronic acid alsoperforms other functions in the biological processes, such as thehydration of tissues, lubrication, cellular migration, cell function anddifferentiation. (See for example A. Balazs et al., Cosmetics &Toiletries, No. 5/84, pages 8-17). Hyaluronic acid may be extracted fromthe above mentioned natural tissues, such as. cocks' combs, or also fromcertain bacteria. Today, hyaluronic acid may also be prepared bymicrobiological methods. The molecular weight of whole hyaluronic acidobtained by extraction is in the region of 8-13 million. However, themolecular chain of the polysaccharide can be degraded quite easily underthe influence of various physical and chemical factors, such asmechanical influences or under the influence of radiation, hydrolyzing,oxydizing or enzymatic agents. For this reason often in the ordinarypurification procedures of original extracts, degraded fractions with alower molecular weight are obtained (See Balazs et al. cited above).Hyaluronic acid, its molecular fractions and the respective salts havebeen used as medicaments and their use is also proposed in cosmetics(see for example the above mentioned article by Balazs et al. and theFrench Patent No. 2478468).

As a therapeutic agent, hyaluronic acid and its salts have been usedespecially in therapy for arthropathies, such as in veterinary medicinefor the cure of arthritis in horses [Acta Vet. Scand. 167, 379 (1976)].As an auxilary and substitutional therapeutic agent for natural tissuesand organs, hyaluronic acid and its molecular fractions and their saltshave been used in ophthalmic surgery (see for example Balazs et al.,Modern Problems in Ophthalmology, Vol. 10, 1970, p. 3--E. B. Strieff, S.Karger eds., Basel; Viscosurgery and the Use of Sodium HyaluronateDuring Intraocular Lens Implantation, Paper presented at theInternational Congress and First Film Festival on IntraocularImplantation, Cannes, 1979; U.S. Pat. No. 4,328,803 with a summary ofthe literature on the uses of HY in ophthalmology; and U.S. Pat. No.4,141,973.

In the application for Italian Patent No. 49 143A83 of Oct. 11, 1983, amolecular fraction of hyaluronic acid is described which can be used,for example as sodium salt, for intraocular and intraarticularinjections suitable for the substitution of internal fluids of the eyeand in arthropathy therapies, respectively. See also co-pending U.S.application Ser. No. 06/756,824 filed on Jul 19, 1985, now abandoned,which is hereby incorporated by reference.

Hyaluronic acid may also be used as an additive for a wide variety ofpolymeric materials used for medical and surgical articles, such aspolyurethanes, polyesters, polyolefins, polyamides, polysiloxanes,vinylic and acrylic polymers and carbon fibers with the effect ofrendering these materials biocompatible. In this case the addition of HYor one of its salts is effected for example by covering the surface ofsuch materials, by dispersion in the same or by both of theseprocedures. Such materials may be used for the manufacture of varioussanitary and medical articles, such as cardiac valves, intraocularlenses, vascular clips, pacemakers and such (see U.S. Pat. No.4,500,676).

Although the term "hyaluronic acid" is commonly used in an impropersense, meaning, as can be seen from above, a whole series ofpolysaccharies with alternations of residues of D-glucuronic acid andN-acetyl-D-glucosamine with varying molecular weights or even degradedfractions of the same, and although the plural form "hyaluronic acids"may seem more appropriate, the discussion herein shall continue to usethe singular form to refer to hyaluronic acid in its various formsincluding its molecular fractions, and the abbreviation "HY" will alsooften be used to describe this collective term.

Relative to the esters of hyaluronic acid, there is a description in theliterature of the methyl ester of a hyaluronic acid with a highmolecular weight obtained by extraction from human umbilical cords[Jeanloz et al., J. Biol. Chem. 186 (1950), 495-511, and Jager et al.,J. Bacteriology 1065-1067 (1979)]. This ester was obtained by treatmentof free hyaluronic acid with diazomethane in ether solution and in itsubstantially all the carboxylic groups proved to be esterified.Furthermore, methyl esters of oligomers of HY with about between 5 and15 disaccharide units have also been described [see Biochem. J. (1977)167, 711-716]. Also described is a methyl ester of hyaluronic acidetherified with methyl alcohol in a part of the hydroxyl alcohol groups[Jeanloz et al., J. Biol. Chem. 194 (1952), 141-150; and Jeanloz et al ,Helvetica Chimica Acta 35 (1952), 262-271]. No biological activity andtherefore no pharmaceutical use has been reported for these esters.

DETAILED DESCRIPTION OF THE INVENTION

According to an object of the present invention the esters of hyaluronicacid with aliphatic, araliphatic, cycloaliphatic or etherocyclicalcohols, in which are esterified all or only a part of the carboxylicgroups of the acid, and the salts of the partial esters with metals orwith organic bases, biocompatible or acceptable from a pharmacologicalpoint of view, possess interesting and precious bio-plastic andpharmaceutical properties and may be used in innumerable fields,including cosmetics, surgery and medicine. In the case of hyaluronicacid, in which the new products qualitatively possess the same orsimilar physical-chemical, pharmacological and therapeutic properties,they are considerably more stable, especially regarding the action ofthe natural enzymes responsible for the degradation of thepolysaccharide molecule in the organism, such as especiallyhyaluronidase, and they, therefore, conserve the above mentionedproperties for very long periods.

A first group of esters according to the present invention, useful intherapy as well as in the other above mentioned fields, is thereforerepresented by those in which the qualities of hyaluronic acid itselfdominate and may be exploited. Such esters derive from alcohols of theabove mentioned series which do not themselves possess a notablepharmacological action, such as for example the saturated alcohols ofthe aliphatic series or simple alcohols of the cycloaliphatic series.

A second group of esters useful in therapy is represented, on the otherhand, by the esters in which the pharmacological qualities of thealcohol component dominate. That is, esters of HY with pharmacologicallyactive alcohols, such as steroid alcohols, such as those of thecortisone type with an antiinflammatory action. These compounds possessproperties qualitatively similar to those of the alcohol, but with amore differentiated range of action, compared also to already knownesters, ensuring a better balanced, constant and regular pharmacologicalaction, and usually obtaining a marked retard effect.

A third group of esters of HY according to the present invention, andwhich represent a particularly original and useful aspect, regards theesters with a more mixed character compared to the two previous groups;That is, esters in which a part of the carboxylic groups of HY areesterified with a pharmacologically active alcohol and another part witha pharmacologically indifferent alcohol, or one whose activity isnegligible. By suitably dosing percentages of the two types of alcoholsas an esterifying component, it is possible to obtain esters with thesame pharmacological activity as the pharmacologically active alcohol,without the specific activity of hyaluronic acid, but having those abovementioned qualities of better stability and bioavailability, withrespect to the activity desired and characteristic of thepharmacologically active alcohol and due to the ester groups of thepharmacologically inert alcohol.

A fourth group of esters is represented by those of a mixed character inwhich the ester groups derive from two different therapeutically activesubstances. In this case also the esters may be partial or total, thatis, only some carboxylic groups are esterified by two differenttherapeutically active alcohols, for example by one cortisone steroidand by an antibiotic or by a phenothiazine, while the carboxylic groupsmay be free or salified, for example with alkaline metals, particularlywith sodium, or all the carboxylic groups are esterified with the abovementioned alcohols. It is, however, also possible to prepare esters withthree or more alcohol components, such as esters in which a part of thecarboxylic groups are esterified with a therapeutically active alcohol,another part with another therapeutically active alcohol, a third partwith a therapeutically inactive alcohol and a fourth part is possiblysalified with a metal and with a therapeutically active or inactive baseor comprises carboxylic groups in a free form.

In the above mentioned esters in which some of the carboxylic acidgroups remain free, these may be salified with metals or organic bases,such as with alkaline or alkaline earth metals or with ammonia ornitrogenous organic bases.

Most of the esters of HY, unlike HY itself, present a certain degree ofsolubility in organic solvents. This solubility depends on thepercentage of esterified carboxylic groups and on the type of alkylgroup linked with the carboxyl. Therefore an HY compound with all itscarboxylic groups esterified presents, at room temperature, goodsolubility for example in dimethylsulfoxide (the benzyl ester of HYdissolves in DMSO in a measure of 200 mg/ml). Most of the total estersof HY present also, unlike HY and especially its salts, poor solubilityin water.

The previously mentioned solubility characteristics, together withparticular and notable viscoelastic properties, allow the use of HYesters to obtain sanitary and medical preparations which are insolublein saline and which have the particular, desired form. These materialsare obtained by preparing a solution of an HY ester in an organicsolvent, forming the very viscose solution into the form of the desiredarticle, and extracting the organic solvent with another solvent whichmixes with the first, but in which the HY ester is insoluble.

The esters of hyaluronic acid according to the present invention are allnew, except for the aforementioned methyl ester of hyaluronic acidextracted from human umbilical cords and the methyl esters of the abovementioned oligomers of HY. Also new, therefore, are partial esters ofhyaluronic acid with methyl alcohol and their salts with metal ororganic bases. The biological and pharmacological activities of theabove mentioned methyl esters described in literature, were unknown, aswere their excellent bioplastic qualities and high stabilty. Alsounknown, therefore, was the use of such esters for the preparation ofmedicaments, cosmetics, sanitary and surgical articles and other newproducts discussed above which are a part of the present invention. Inparticular therefore, the pharmaceutical preparations containing thealready known methyl esters of HY are also new.

Finally, the present invention also includes the use of the abovementioned new products and of the above mentioned known methyl ester, asvehicles for active pharmaceutical substances, and medicaments includingthe above mentioned esterified derivatives of hyaluronic acid and one ormore of such active substances. In such medicaments the hyaluronic esteror one of its salts is preferably derived from pharmacologicallyinactive alcohols, but may also represent an ester derived from apharmacologically active alcohol and have itself a pharmacologicalactivity. The vehicling action of the component represented by thehyaluronic ester or one of its salts is to be observed in a moresatisfactory assimilation of the active substance in conditions whichare particularly compatible with the biological environment of the organto be treated. This is especially the case in the field ofophthalmology. Of these medicaments containing a hyaluronic ester asvehicle, those for topical use are particularly important. The mainobject of the present invention is, therefore, represented by the totalor partial esters of hyaluronic acid with alcohols of the aliphatic,araliphatic, cycloaliphatic or heterocyclic series and by the salts ofsuch partial esters with inorganic or organic bases, with the exceptionof the methyl ester of hyaluronic acid in which substantially all thecarboxylic groups are esterified.

A second object of the present invention is represented by thepharmaceutical preparations containing as their active ingredient one ormore esters of hyaluronic acid or one of their salts as described above,including the total methyl ester of hyaluronic acid, and the use of suchesters for therapeutic uses.

A third object of the present invention is represented by medicamentsincluding:

1) a pharmacologically active substance or an association ofpharmacologically active substances and

2) a carrying vehicle made up of a total or partial ester of hyaluronicacid with alcohols of the aliphatic, araliphatic, cycloaliphatic orheterocyclic series or of the salts of such partial esters withinorganic or organic bases, with the addition, if desired, of hyaluronicacid or one of its salts with inorganic or organic bases, and by the useof such medicaments for therapeutic uses.

A fourth object of the present invention is represented by the use ofthe esters and their salts as described above in cosmetics, and bycosmetic articles containing such esters.

A fifth object of the present invention is represented by the use of theesters and their salts as described above for the manufacture ofsanitary and surgical plastic articles and by these articles themselves.

Another use of the new and the known esters according to the inventionrefers to the manufacture of sanitary, cosmetic and surgical articles inwhich the hyaluronic esters act as vehicles for the basic substanceswhich serve this purpose, such as the various polymers mentioned above.

Another object of the invention is finally represented by the proceduresfor the preparation of the esters of hyaluronic acid and of the salts ofthe partial esters.

Alcohols of the aliphatic series to be used as esterifying components ofthe carboxylic groups of hyaluronic acid according to the presentinvention are for example those with a maximum of 34 carbon atoms, whichmay be saturated or unsaturated and which may possibly also besubstituted by other free functional or functionally modified groups,such as amine, hydroxyl, aldehyde, ketone, mercaptan, or carboxyl groupsor by groups derived from these, such as hydrocarbyl ordi-hydrocarbylamine groups (from now on the term "hydrocarbyl" will beused to refer not only to monovalent radicals of hydrocarbons such asthe C_(n) H_(2n+1) type, but also bivalent or trivalent radicals, suchas "alkylenes" C_(n) H_(2n) or "alkylidenes" C_(n) H_(2n)), ether orester groups, acetal or ketal groups, thioether or thioester groups, andesterified carboxyl or carbamide groups and carbamide substituted by oneor more hydrocarbyl groups, by nitrile groups or by halogens.

Of the above mentioned groups containing hydrocarbyl radicals, these arepreferably lower aliphatic radicals, such as alkyls, with a maximum of 6carbon atoms. Such alcohols may also be interrupted in the carbon atomchain by heteroatoms, such as oxygen, nitrogen and sulfur atoms.Preferred are alcohols substituted with one or two of the saidfunctional groups.

Alcohols of the above mentioned group which are preferably to be usedwithin the bounds of the present invention are those with a maximum of12, and especially 6 carbon atoms, and in which the hydrocarbyl atoms inthe above mentioned amine, ether, ester, thioether, thioester, acetal,ketal groups represent alkyl groups with a maximum of 4 carbon atoms,and also in the esterified carboxyl or substituted carbamide groups thehydrocarbyl groups are alkyls with the same number of carbon atoms, andin which in the amine or carbamide groups may be alkylenamine oralkylencarbamide groups with a maximum of 8 carbon atoms. Of thesealcohols special mention should be given to those which are saturatedand not substituted such as the methyl, ethyl, propyl, and isopropylalcohols, normal butyl alcohol, isobutyl alcohol, tertiary butylalcohol, the amyl, pentyl, hexyl, octyl, nonyl and dodecyl alcohols and,above all, those with a linear chain, such as normal octyl and dodecylalcohols. Of the substituted alcohols of this group, the bivalentalcohols should be listed, such as ethyleneglycol, propyleneglycol andbutyleneglycol, the trivalent alcohols such as glycerine, the aldehydealcohols such as tartronic alcohol, the carboxylic alcohols such aslactic acids, for example glycolic acid, malic acid, the tartaric acids,citric acid, the aminoalcohols, such as normal aminoethanol,aminopropanol, normal aminobutanol and their dimethylated anddiethylated derivatives in the amine function, choline,pyrrolidinylethanol, piperidinylethanol, piperazineylethanol and thecorresponding derivatives of normal propyl or normal butyl alcohol,monothioethyleneglycol or its alkyl derivatives, such as the ethylderivative in the mercaptan function.

Of the higher saturated aliphatic alcohols the following should bementioned: cetyl alcohol and myricyl alcohol, but for the aim of thepresent invention the higher unsaturated alcohols with one or two doublebonds, are especially important, such as especially those contained inmany essential oils and with affinity to terpene, such as citronellol,geraniol, nerol, nerolidol, linalool, farnesol, phytol. Of theunsaturated lower alcohols it is necessary to consider allyl alcohol andpropargyl alcohol. Of the araliphatic alcohols special attention shouldbe given to those with only one benzene residue and in which thealiphatic chain has a maximum of 4 carbon atoms, which the benzeneresidue can be substituted by between 1 and 3 methyl or hydroxyl groupsor by halogen atoms, especially by chlorine, bromine and iodine, and inwhich the aliphatic chain may be substituted by one or more functionschosen from the group containing fee amine groups or mono- ordimethylated or by pyrrolidine or piperidine groups. Of these alcoholsspecial attention should be given to benzyl alcohol and phenetylalcohol.

The alcohols of the cycloaliphatic or aliphatic-cycloaliphatic seriesmay derive from mono- or polycyclic hydrocarbons, may preferably have amaximum of 34 carbon atoms, may be unsubstituted and may contain one ormore substituents, such as those mentioned above for the aliphaticalcohols. Of the alcohols derived from cyclic monoannular hydrocarbons,special mention should be given to those with a maximum of 12 carbonatoms, the rings with preferably between 5 and 7 carbon atoms, which maybe substituted for example by between one and three lower alkyl groups,such as methyl, ethyl, propyl or isopropyl groups. As specific alcoholsof this group the following can be mentioned: cyclohexanol,cyclohexanediol, 1,2,3 cyclohexanetroil and 1,3,5 cyclohexanetriol(phloroglucitol), inositol, and the alcohols which derive from p-methanesuch as carvomenthol, menthol, and α- γ terpineol, 1-terpineol,4-terpineol and piperitol, or the mixture of these alcohols known as"terpineol", 1,4- and 1,8 terpin. Of the alcohols which derive fromhydrocarbons with condensed rings, such as those of the thujane, pinaneor comphane, the following can be mentioned: thujanol, sabinol, pinolhydrate, D and L-borneol and D and L-isoborneol.

Aliphatic-cycloaliphatic polycyclic alcohols to be used for the estersof the present invention are sterols, cholic acids and steroids, such assexual hormones and their synthetic analogues, especiallycorticosteroids and their derivatives. It is therefore possible to use:cholesterol, dihydrocholesterol, epidihydrocholesterol, coprostanol,epicoprostanol, sitosterol, stigmasterol, ergosterol, cholic acid,deoxycholic acid, lithocholic acid, estriol, estradiol, equilenin,equilin and their alkylate derivatives, as well as their ethynyl orpropynyl derivatives in position 17, such as 17 α-ethynlestradiol or 7α-methyl-17 α-ethynyl-estradiol, pregnenolone, pregnanediol,testosterone and its derivatives, such as 17 α-methyltestosterone,1,2-dehydrotestosterone and 17 α-methyl-1,2-dehydrotesterone, thealkynylate derivatives in position 17 of testosterone and1,2-dehydrotestosterone, such as 17 α-ethynyltestosterone, 17α-propynyltestosterone, norgestrel, hydroxyprogesterone, corticosterone,deoxycorticosterone, 19-nortestosterone, 19-nor-17 α-methyltestosteroneand 19-nor-17 α-ethynyltestosterone, antihormones such as cyproterone,cortisone, hydrocortisone , prednisone , prednisolone , fluorocortisone,dexamethasone, betamethansone, paramethasone, flumethasone,fluocinolone, fluprednylidene, clobetasol, beclomethasone, aldosterone,deoxycorticosterone, alfaxolone, alfadolone, bolasterone. As esterifyingcomponents for the esters of the present invention the following areuseful: genins (aglycons) of the cardioactive glucosides, such asdigitoxigenin, gitoxigenin, digoxigenin, strophanthidin, tigogenin andsaponins.

Other alcohols to be used according to the invention are the vitaminones, such as axerophthol, vitamins D₂ and D₃, aneurine, lactoflavine,ascorbic acid, riboflavine, thiamine, pantothenic acid.

Of the heterocyclic acids the following can be considered as derivativesof the above mentioned cycloaliphatic or aliphatic-cycloaliphaticalcohols if their linear or cyclic chains are interrupted by one ormore, for example by between one and three heteroatoms, for instancechosen from the group formed by --O--, --S--, --N, --NH-- and in thesethere may be one or more unsaturated bonds, for example double bonds, inparticular between one and three, thus including also heterocycliccompounds with aromatic structures. For example the following should bementioned: furfuryl alcohol, alkaloids and derivatives such as atropine,scopolamine, cinchonine, la cinchonidine, quinine, morphine, codeine,nalorphine, N-butylscopolammonium bromide, ajmaline; phenylethylaminessuch as ephedrine, isoproterenol, epinephrine; phenothiazine drugs suchas perphenazine, pipothiazine, carphenazine, homofenazine,acetophenazine, fluophenazine, N-hydroxyethylpromethazine chloride;thioxanthene drugs such as flupenthixol and clopenthixol;anticonvulsants such as meprophendiol; antipsychotics such as opipramol;antiemetics such as oxypendyl; analgesics such as carbetidine andphenoperidine and methadol; hypnotics such as etodroxizine; anorexicssuch as benzidrol and diphemethoxidine; minor tranquilizers such ashydroxyzine; muscle relaxants such as cinnamedrine, diphylline,mephenesin, methocarbamol, chlorphenesin, 2,2-diethyl-1,3-propanediol,guaifenesin, hydrocilamide; coronary vasodilators such as dipyridamoleand oxyfedrine; adrenergic blockers such as propanolol, timolol,pindolol, bupranolol, atenolol, metoprolol, practolol; antineoplasticssuch as 6-azauridine, cytarabine, floxuridine; antibiotics such aschloramphenicol, thiamphenicol, erythromycin, oleandomycin, lincomycin;antivirals such as idoxuridine; peripheral vasodilators such asisonicotinyl alcohol; carbonic anhydrase inhibitors such assulocarbilate; antiasthmatic and antiinflammatories such as tiaramide;sulfamidics such as 2-p-sulfanilonoethanol.

As discussed above, in some cases hyaluronic acid esters may be ofinterest where the ester groups derive from two or more therapeuticallyactive hydroxylic substances, and naturally all possible variants may beobtained. Especially interesting are the substances in which two typesof different ester groups deriving from drugs of a hydroxylic characterare present and in which the remaining carboxyl groups are free,salified with metals or with one or various bases listed later, possiblyalso bases being themselves therapeutically active, for example with thesame or similar activity as that of the esterifying component. Inparticular, it is possible to have hyaluronic esters deriving on the onehand from an antiinflammatory steroid, such as one of those mentionedpreviously, and on the other hand from a vitamin, from an alkaloid orfrom an antibiotic, such as one of those listed.

The degree of esterification of hyaluronic acid with the above mentionedalcohols depends firstly on the special properties to be obtained in thevarious fields of application, for example a greater or lesserlipophilia or hydrophilia with respect to certain tissues such as theskin.

Normally, a high degree of esterification up to total esterification ofhyaluronic acid increases its lipophilic character and therefore lessensits solubility in water. For a therapeutic use of the new esters of thisinvention, for example, it is especially important to regulate thedegree of esterification in order to ensure, despite a good andincreased level of lipophilia compared to hyaluronic acid or its sodiumsalt, sufficient hydrosolublity, for example, a solubility of 10 mg/ml.Naturally it is necessary to bear in mind the influence of the molecularsize of the same esterifying component, which usually influenceshydrosolubility in an inversely proportional manner. As has already beensaid, the esterification of the carboxylic groups of hyaluronic acid mayplay various roles, which may be useful in different fields, for examplein medicine using the esters as therapeutic agents or in surgery usingthem as plastic articles. For use in therapy, it has already been saidthat it is possible to consider the esterification of an alcohol whichis itself therapeutically active, such as an antiinflammatorycortisteroid, with hyaluronic acid as a means to improve its therapeuticefficacy.

Regarding similar therapeutically active alcohols, hyaluronic acid actstherefore as a particularly efficient vehicle which is perfectlycompatible with the biological environment. In the above list ofalcohols to be used for esterification according to the presentinvention, there appear several of these pharmacologically activealcohols and therefore the possible applications of the correspondingesters are apparent as the indications are the same as those for thefree alcohols. Again, as has already been said, in partial esters withtherapeutically active alcohols it is possible to esterify part or allof the remaining carboxylic groups of the hyaluronic component withpharmacologically inert alcohol, such as the saturated lower aliphaticalcohols, such as ethyl or isopropyl alcohol.

A particularly interesting aspect of the present invention is thepossibility of preparing more stable drugs than those available atpresent. It is possible therefore on the one hand to prepare esters ofhyaluronic acid with therapeutically inactive alcohols for use intypical indications of hyaluronic acid itself, such as forintra-articular injections, where the ester acts as a lubricant. Due tothe improved stability of the esters relative to hyaluronidase ascompared to the free acid, it is possible to obtain quite a considerableprolonged action. On the other hand, it is possible to obtain drugs witha "retard" actions for the above-mentioned esters of HY withtherapeutically active alcohols, possibly also salified withtherapeutically active bases. The liberation of the active alcohols dueto esterase and that of the salified groups due to the hydrolitic actionis very slow.

For cosmetic use it is preferable to use total or partial esters ofhyaluronic acid with pharmacologically inert alcohols, such as saturatedor unsaturated aliphatic alcohols, for example non-substituted alcoholsof this type with a straight or ramified chain, for example with between1 and 8 carbon atoms, such as those specifically mentioned previously.Particularly interesting are those unsaturated alcohols, such as withone or more double bonds, such as vinyl or allyl alcohols and theircondensed derivatives, such as especially polyvinyl alcohols orpolyvalent alcohols, such as glycerine. In this case also it is possibleto use, according to the intended purpose, mixed esters.

Also useful are cycloaliphatic alcohols, such as derivatives ofcyclopentane or cyclohexane and their derivatives substituted by loweralkyl groups, such as alkyls with between 1 and 4 carbon atoms,especially by methyl groups. Of particular interest are also esters withcycloaliphatic and aliphatic alcohols--cycloaliphatics derived fromterpene, such as those mentioned above and from therapeutically activealcohols, and which may also be used in cosmetics.

The alcohols to be used preferably to make articles for sanitary andsurgical use are essentially the same as those listed above for cosmeticuse. In the partial esters according to the invention the percentage ofesterified groups may vary greatly in relation to the use for which theproduct is intended, and that is above all with regard to the use in thevarious fields of application mentioned above.

Of particular interest however are those partial esters in which atleast 5% and at most 90% of all the carboxylic groups of HY areesterified, and especially those with an esterified percentage ofbetween 50 and 80%.

The ratio between the number of different types of ester groups mayobviously also vary in the mixed partial esters. For example in the caseof two types of such groups, the ratio varies preferably between 0:1:1and 1:01, and the same is true of total esters. For the esters intendedfor therapeutic use the ratio varies preferably between 0.5:1 and 1:0.5.Such ratios are preferably also valid for total esters and, in thepartial esters, they are to be taken preferably with reference to thepercentages mentioned above regarding the inclusive number of esterifiedgroups.

In the partial esters of the invention the non-esterified carboxylicgroups may be kept free or may be salified. For the formation of suchsalts the bases are chosen according to the criterion of these for whichthe product is intended. It is possible to form inorganic salts derivingfrom alkaline metals, such as potassium and especially sodium andammonium, or deriving from alkaline earth metals, such as calcium, ormagnesium or aluminum salts.

Particularly interesting are the salts with organic bases, especiallynitrogenized bases and therefore aliphatic, arylaliphatic,cycloaliphatic or heterocyclic amines.

These ammonic salts may derive from therapeutically acceptable butinactive amines or from amines with therapeutic action. Of the formerthe aliphatic amines above all should be considered, such as mono- di-and tri-alkylamines with alkyl groups having a maximum of 18 carbonatoms or arylalkylamines with the same number of carbon atoms in thealiphatic part and where aryl means a benzene group possibly substitutedby 1 and 3 methyl groups or halogen atoms or hydroxyl groups. Thebiologically inactive bases for the formation of salts may also becyclic such as monocyclic alkylenamines with cycles of between 4 and 6carbon atoms, possibly interrupted in the cycle by heteroatoms chosenfrom the group formed by nitrogen, oxygen and sulfur, such as piperidineor morpholine, and may be substituted for example by aminic orhydroxylic functions, such as aminoethanol, ethylendiamine, ephedrine orcholine.

It is also possible to form the quarternary ammonium salts of thepartial esters, for example the salts of tetraalkylammonium with theabove mentioned number of carbon atoms and preferably salts of such atype in which the fourth alkyl group has between 1 and 4 carbon atoms,for example a methyl group.

Among the biologically active amines whose therapeutic actions may beput to use, are included all the nitrogenized and basic drugs such asthose included in the following groups: alkaloids, peptides,phenothiazines, benzodiazepines, thioxanthenes, hormones, vitamins,anticonvulsants, antipsychotics, antiemetics, anesthetics, hypnotics,anorexics, transquilizers, muscle relaxants, coronary vaso dilators,antineoplastics, antibiotics, antibacterials, antivirals, antimalarials,carbonic anhydrase inhibitors, non-steroid antiinflammatory agents,vasoconstrictors, cholinergic agonists, cholinergic antagonists,adrenergic agonists, adrenergic antagonists, narcotic antagonists.

All those drugs with basic nitrogenized groups listed in the inventionand regarding the use of the esters may be quoted as examples.

According to a particular aspect of the present invention the newhyaluronic esters and their salts may be used as an excellent vehiclefor therapeutically active substances. To this end it is possible to usethe total esters or the partial esters of the salified partial esters inthe remaining carboxylic groups, for example with one of the abovementioned substances therapeutically acceptable but not biologicallyactive, above all with alkaline metals, for example sodium. These arethe above mentioned medicaments made by association containing twocomponents:

Component (1)--a pharmacologically active substance or an association oftwo or more active substances; and

Component (2)--a carrying vehicle comprising a partial or total ester ofhyaluronic acid with an alcohol, or the salts of such partial esterswith an organic or inorganic base, optionally with the addition ofhyaluronic acid or a salt thereof with an inorganic or organic base.

The hyaluronic esters to be used in these medicaments are above allthose in which the esterifying alcohol is not itself pharmacologicallyactive, for example a simple aliphatic alcohol, as described above.Medicaments of this type in which the ester also is pharmacologicallyactive, for example a simple aliphatic alcohol, as described above.Medicaments of this type in which the ester also is pharmacologicallyactive are not excluded from this aspect of the invention, as forexample in the case of one of the esters described above deriving fromalcohols with pharmacological action.

In the same way, the invention also includes medicaments of this type inwhich the esters of Component (2) are salified also with therapeuticallyactive bases. These bases may be the same pharmacologically activesubstances vehicled in the hyaluronic ester, and the mixture in thiscase, as described below, therefore contains salts of a partial ester ofhyaluronic acid with therapeutically active bases, possibly in thepresence of an excess of active base component (1). The case may on theother hand present itself where the vehicled substance is not of a basicnature, and free carboxylic groups in the hyaluronic ester are stillsalified with therapeutically active bases.

The use of hyaluronic esters as a vehicle allows therefore thepreparation of the new medicaments described above, including 1) apharmacologically active substance or an association of two or more ofsuch substances and 2) a hyaluronic ester as described above or one ofits salts, and such medicaments are a further object of the invention.In such medicaments, if partial esters of HY are used, the possiblesalification of the remaining carboxylic groups is carried outpreferably with therapeutically neutral inorganic or organic bases,especially with alkaline metals such as sodium or ammonium. Should theactive substance component (1) or a corresponding association ofsubstances have basic groups, such as for example antibiotics containingamine groups, and if partial esters of hyaluronic acid should be usedwith remaining free carboxylic groups, the corresponding salts areformed between the carboxylic groups and these basic substances. The newmedicaments therefore include in particular partial esters of hyaluronicacid partially and totally salified with pharmacologically activesubstances and of a basic character. As described above, particularlyimportant are the associated medicaments of the type described here, inwhich Component (1) is a pharmacologically active substance for topicaluse.

The use of hyaluronic esters as a vehicle for drugs to be appliedtopically is particularly useful in ophthalmology where a particularcompatibility is to be observed for the new products with the cornealepithelium, and therefore excellent tolerability, without anysensitization effects. Furthermore, when the medicaments areadministered in the form of concentrated solutions with elastic-viscosecharacteristics or in solid form, it is possible to achieve homogenousand stable films which are perfectly transparent and adhesive on thecorneal epithelium, guaranteeing prolonged bioavailability of the drugand therefore representing excellent preparations with a retard effect.

Such ophthalmic medicaments are particularly valuable in the veterinaryfield, considering for example that at present there are no veterinaryspecialities for oculistic use containing chemotherapeutic agents.Indeed, preparations intended for human use are usually used, and thesedo not always guarantee a specific range of action or they do not makeallowances for the particular conditions in which the treatment musttake place. This, for example, is the case in therapy for infectivekeratoconjunctivities, pink eye or IBK, an infection which usuallyaffects cattle, sheep and goats. Presumably for these three speciesthere are specific etiological factors and more particularly: in cattlethe main microorganism involved seems to be Moraxella bovis (even thoughother agents of a viral origin should not be excluded, such as forexample Rinotracheitis virus, in sheep Micoplasma, Rickettsiae andClamidiae, and in goats Rickettsiae). The disease manifests itself inacute form and tends to spread rapidly: in the initial stages thesymptomatology is characterized by blepharospasm and excessivelachrymtion, followed by purulent exudate, conjunctivities andkeratitis, often accompanied by fever, loss of appetite and milkproduction. Particularly serious are the corneal lesions which in thefinal stages may even cause perforation of the cornea itself. Theclinical progress of the disease varies from a few days to severalweeks.

A vast selection of chemotherapeutic agents are used for treatment,administered both topically (often in association with steroidantiinflammatory agents), and systemic, including: tetracyclines, suchas oxytetracycline, penicillins, such as cloxacillin andbenzylpenicillin, sulfonamides, polymyxin B (associated with miconazoleand prednisolone), chloramphenicol, tylosin and chloromycetin. Topicaltreatment of the disease, despite its apparent simplicity, is still anunsolved problem, since with the oculistic preparations used so far, ithas not been possible for one reason or another, to obtaintherapeutically efficient concentrations of antibiotics or sulphamidesin the lachrymal secretion. This is quite understandable in the case ofsolutions, considering the mainly inclined position of the head in theseanimals, but the same is also true of semisolid medicaments, as thecommonly used excipients do not possess the necessary qualities ofadhesiveness to the corneal surface, since they do not usually have ahigh enought concentration of active substance and cannot achieveperfect distribution of the same (i.e., there is a presence of adistribution gradient). These defects of conventional colliriums inophthalmic use have for example been described by Slatter et al., Austr.Vet. J., 1982, 59 (3), pp. 69-72.

With the esters of the present invention these difficulties can beovercome. The presence of the hyaluronic acid ester as a vehicle forophthalmic drugs in fact allows the formulation of excellentpreparations with no concentration gradients of the active substance andthey are therefore perfectly homogenous, with perfect transparency andexcellent adhesiveness to the corneal epithelium, with no sensitizationeffects, with excellent vehicling of the active substance and possibly aretard effect.

The above mentioned properties of the new medicaments may of course beexploited also in fields other than ophthalmology. They may be used indermatology and in diseases of the mucous membranes, for example in themouth. Furthermore, they may be used to obtain a systemic effect due tothe effect of transcutaneous absorption, such as in suppositories. Allthese applications are possible both in human and veterinary medicine.In human medicine the new medicaments are particularly suitable for usein pediatrics. The present invention includes therefore in particularany of these therapeutic applications.

For the sake of brevity, from now on when the active substance ofcomponent (1) according to the invention is mentioned, it is to beunderstood to also include the association of one or more activesubstances.

The component (1) described above may first of all be defined in regardto its use in the various fields of therapy, starting with thedistinction between human and veterinary medicine, and then specifyingthe various sectors of application with regard to the organs or tissuesto be treated, such as, with reference to topical use, ophthalmology,dermatology, otorhinolaryngology, gynecology, angiology, neurology orany type of pathology of internal organs which may be treated withtopical applications, for example with rectal applications.

The vehicling action of the hyaluronic esters also applies to associatedmedicaments of the type mentioned above in which the active substanceacts not only topically or by nasal or rectal absorption, for example bynasal sprays or preparations for inhalation for the oral cavity or thepharynx, but also by oral or parenteral route, for example byintramuscular, subcutaneous or intravenous route, as it favorsabsorption of the drug into the application site. The new medicamentscan therefore be applied, apart from in the fields already mentioned, inpractically all sectors of medicine, such as internal medicine, forexample in pathologies of the cardiovascular system, in infections ofthe respiratory system, the digestive system, the renal system, indiseases of an endocrinological nature, in oncology, in psychiatry etc.,and may also be classified therefore from the point of view of theirspecific action, being perhaps anesthetics, analgesics,antiinflammatories, wound healers, antimicrobics, adrenergic agonistsand antagonists, cytostatics, antirheumatics, antihypertensives,diuretics, sexual hormones, immunostimulants and immunosuppressants, forexample, one of the drugs having the activity already described for thetherapeutically active alcohols to be used as esterifying componentaccording to the present invention, or for the therapeutically activebases used for the salification of the free carboxylic groups.

Component (1) of the above mentioned medicaments may also be, accordingto the invention, an association of two or more active substances, ascontained in many known medicaments.

Regarding the field of ophthalmology, the indications may be forexample: the miotic, antiinflammatory, wound healing and antimicrobialeffects.

Examples of pharmacologically active substances to be used in ophthalmicmedicaments according to the invention are: basic and non-basicantibiotics such as aminoglycosides, macrolides, tetracyclines andpeptides, such as gentamycin, neomycin, streptomycin,dihydrostreptomycin, kanamycin, amikacin, tobramycin, spectinomycin,erythromycin, oleandomycin, carbomycin, spiramycin, oxytetracycline,rolitetracycline, bacitracin, polymyxin B. gramicidin, colistin,chlkoramphenicol, lincomycin, vancomycin, novobiocin, ristocetin,clindamycin, amphotericin B, griseofulvin, nystatin, and possibly theirsalts such as sulfate or nitrate, or associations of the same betweenthemselves or with other active ingredients, such as those mentionedbelow.

Other ophthalmic drugs to be used to advantage according to the presentinvention are: other antiinfectives such as diethylcarbamazine,mebendazole, sulfamidics such as sulfacetamide, sulfadiazine,sulfisoxazole, antivirals and antitumorals such as iododeoxyuridine,adenine arabinoside, trifluorothymidine, acyclovir, ethyldeoxyuridine,bromovinyldeoxyuridine, 5-iodo-5'-amino-2',5'-dideoxyuridine; steroidantiinflammatories, such as dexamethasone, hydrocortisone, prednisolone,fluorometholone, medrysone and possibly their esters, for examplephosphoric acid; non-steroid antiinflammatories such as indomethacin,oxyphenbutazone, flurbiprofen; wound healers such as epidermal growthfactor, EGF; local anesthetics, such as Benoxinate, proparacaine andpossibly their salts; cholinergic agonists such as pilocarpine,methcholine, carbomylcholine, aceclidine, physostigmine, neostigmine,demecarium and possibly their salts; cholinergic antagonist drugs suchas atropine and their salts; adrenergic agonist drugs such asnoradrenaline, adrenaline, naphazoline, methoxamine and possibly theirsalts; adrenergic antagonist drugs such as propanolol, timolol,pindolol, bupranolol, atenolol, metoprolol, oxprenolol, practolol,butoxamine, sotalol, butathrin, labetolol and possibly their salts.

Examples of the active substances to be used alone or in associationamong themselves or with other active principles in dermatology are:therapeutic agents as antiinfective agents, antibiotics, antimicrobials,antiinflammatory, cytostatic, cytotoxic, antiviral, anesthetic agents,and prophylactic agents, such a sun screens, deodorants, antiseptics anddisinfectants. Of the antibiotics, particularly important are:erythromycin, bacitracin, gentamicin, neomycin, aureomicin, gramicidinand their associations, of the antibacterials and disinfectants:nitroflurzone, mafenide, chlorhexidine, and derivatives of81-hydroxyquinoline and possibly their salts; of the antiinflammatoryagents above all the corticosteroids such as prednisolone,dexamethazone, flumethasone, clobetasol, triamcinolone acetonide,betamethasone and their esters, such a valerates, benzoates,dipropionates; of the cytotoxic group,: fluorouracil, methotrexate,pdophyllin; of the anesthetics dibucaine, lidocain, benzocaine.

This list of course only gives some examples and any other agentsdescribed in the literature may be used.

As associations of drugs to be used in dermatology, the variousantibiotics should be mentioned, such as erythromycin, gentamycin,neomycin, gramicidin, polymyxin B, among themselves, or associations ofthese antibiotics with antiinflammatory agents, for examplecorticosteroids, for example hydrocortisone+neomycin,hydrocortisone+neomycin+polymyxin B+gramicidin, dexamethasone+neomycin,fluorometholone+neomycin, prednisolone+neomycin,triamcinolone+neomycin+gramicidin+nystatin, or any other associationused in conventional preparations for dermatology.

The associations of various active substances are not of course limitedto this field, but in each of the above mentioned sectors of medicine itis possible to use associations similar to those already in use for theknown pharmaceutical preparations of the art.

In the above case of the use of a component (1) of a basic character,the salts which are formed with a partial hyaluronic ester (since thelatter is used to excess) may be of various types, that is, all theremaining carboxylic groups may be salified or only an aliquot part,thereby producing esters--acid salts, or esters--neutral salts. Thenumber of acid groups which are to be kept free may be of importance forthe preparation of medicaments with a particular pH. Vice versa, it ispossible to use an excess of basic component (1), in which case all thecarboxylic groups available in the hyaluronic ester are salified withthe base.

According to a particular aspect of the invention it is possible toprepare the medicaments of this type starting from previously isolatedand possibly purified salts, in their solid anhydrous state, asamorphous powders, which form an aqueous solution on contact with thetissue to be treated, characterized by viscosity and elastic properties.These qualities are maintained even at stronger dilutions and it ispossible therefore to use, in the place of the above mentioned anhydroussalts, more or less concentrated solutions in water or saline, possiblywith the addition of other excipients or additives, such as for exampleother mineral salts to regulate the pH and osmotic pressure. It is ofcourse possible to use the salts also for the preparation of gels,inserts, creams or ointments, containing also other excipients oringredients used in traditional formulations of these pharmaceuticalpreparations.

According to a major aspect of the invention however, the medicamentscontaining the hyaluronic ester or their salts with therapeuticallyactive or inactive substances as a vehicle are used alone (exceptpossibly with an aqueous solvent). Also included in the invention arethe mixtures obtainable from all the types of medicaments describedhere, mixtures of the same medicaments, and also possibly mixtures ofthe new hyaluronic esters with free hyaluronic acid or mixtures of theirsalts, for example sodium salts.

Component (1) according to the invention may also be associations ormixtures of two or more such drugs and possibly also with otherprinciples. For example, in ophthalmology, a drug may be associated withan antibiotic or antiphlogistic substance and a vasoconstrictor or withseveral antibiotics, one or more antiphlogistic substances, or with oneor more antibiotics, a mydiatric or a miotic or wound healing orantiallergic agent, etc. For example the following associations ofophthalmic drugs may be used: kanamycin phenylephrine+dexamethasonephosphate; kanamycin+betamethasone phosphate+phenylephrine; or similarassociations with other antibiotics used in ophthalmology, such asrolitetracycline, neomycin, gentamicin, tetracycline.

If in the place of just one active substance component (1), associationsof active substances are used, such as those mentioned above, the saltsof the basic active substances and the partial ester of hyaluronic acidmay be mixed salts of one or more of such basic substances or possiblymixed salts of this type with a certain number of other acid groups ofthe polysaccharides salified with metals or bases mentioned above. Forexample, it is possible to prepare salts of a partial ester ofhyaluronic acid or of one of the molecular fractions Hyalastine orHyalectin with a pharmacologically inactive alcohol, for example a loweralkanol and with a certain percentage of salified acid groups with theantibiotic kanamycin, another percentage of carboxylic groups salifiedwith the vasoconstrictor phenylephrine, and a remaining percentage ofacid groups may be, for example, free of salified with sodium or one ofthe other above mentioned metals. It is also possible to mix this typeof mixed salt with free hyaluronic acid or its fractions or theirmetallic salts, as indicated above for the medicaments containing saltsof one single active substance with the aforementioned polysaccharideesters.

Of the examples discussed for ophthalmology and dermatology it ispossible to understand by analogy which medicaments according to thepresent invention are to be used in the above mentioned fields ofmedicine, such as for example in otorhinolaryngology, odontology or ininternal medicine, for example in endocrinology. Such preparations may,therefore, be for example antiinflammatories, vasoconstrictors, orvasocompressors such as those already mentioned for ophthalmology,vitamins, antibiotics, such as those mentioned above, hormones,chemiotherapics, antibacterials, etc. also as mentioned above for use indermatology.

The associated medicaments of a hyaluronic ester with apharmacologically active substance may contain other pharmaceuticalvehicles, such as those mentioned below for the pharmaceuticalpreparations containing only hyaluronic esters, and may appear in theform of ointments, creams, pastilles, gelatine capsules, capsules,aqueous or oily solutions, sprays, suppositories, etc. However,according to a particular aspect of the present invention it ispreferable to use medicaments containing an association of components(1) and (2), with component (2) as the sole vehicle (apart from apossible solvent such as an aqueous solvent).

Of the medicaments of the invention the following are of particularimportance, according to each case, those with a degree of aciditysuitable for the environment to which they are to be applied, that iswith a physiologically tolerable pH. The adjustment of the pH, forexample in the above mentioned salts of the partial ester of hyaluronicacid with a basic active substance, may be done by suitably regulatingthe quantities of polysaccharide, of its salts and of the basicsubstance itself. Thus, for example, if the aciditiy of a salt of thepartial ester of hyaluronic acid with a basic substance is too high, theexcess of free acid groups cans be neutralized with the above mentionedinorganic bases, for example with the hydrate of sodium or potassium orammonium.

Method of Preparing HY Esters of the Invention Method A

The esters of hyaluronic acid according to the invention may be preparedby methods known per se for the esterification of carboxylic acids, forexample by treatment of free hyaluronic acid with the desired alcoholsin the presence of catalyzing substances, such as strong inorganic acidsor ionic exchangers of the acid type, or with an etherifying agentcapable of introducing the desired alcoholic residue in the presence ofinorganic or organic bases. As etherifying agents it is possible to usethose known in literature, such as especially the esters of variousinorganic acids or of organic sulphonic acids, such as hydracids, thatis hydrocarbyl halogenides, such as methyl or ethyl iodide, or neutralsulphates or hydrocarbyl acids, alfites, carbonates, silicates,phosphites or hydrocarbyl sulfonates, such as methyl benzene orp-toluenesulfonate or methyl or ethyl chlorosulfonate. The reaction maytake place in a suitable solvent, for example an alcohol, preferablythat corresponding to the alkyl group to be introduced in the carboxylgroup. But the reaction may also take place in non-polar solvents, suchas ketones, ethers, such as dioxane or aprotic solvents, such asdimethylsulphoxide. As a base it is possible to use for example ahydrate of an alkaline or alkaline earth metal or magnesium or silveroxide or a basic salt or one of these metals, such as a carbonate, and,of the organic bases, a tertiary azotized base, such as pyridine orcollidine. In the place of the base it is also possible to use an ionicexchanger of the basic type.

Another esterification method employs the metal salts or salts withorganic azotized bases, for example ammonium or ammonium substitutesalts. Preferably, the salts of the alkaline or alkaline earth metalsare used, but also any other metallic salt may be used. The esterifyingagents are also in this case those mentioned above and the same appliesto the solvents. It is preferable to use aprotic solvents, for exampledimethylsulphoxide and dimethylformamide.

In the esters obtained according to this procedure or according to theother procedure described hereafter, free carboxylic groups of thepartial esters may be salified, if desired, in a per se known manner.

Method B

The hyaluronic esters of the present invention may, however, be preparedto advantage according to a second method which may be generally appliedto the preparation of carboxylic esters of acidic polysaccharides withcarboxyl groups. This method consists of treating a quaternary ammoniuimsalt of an acidic polysaccharide containing carboxyl groups with anetherifying agent, preferably in an aprotic organic solvent. As startingacidic polysaccharides it is possible to use, for example, apart fromhyaluronic acid, other acidic polysacchariddes of animal or vegetableorigin and synthetically modified derivatives of the same, such as acidhemicellulose, obtainable from the alkaline extracts of certain plantsand after precipitation of xylans, whose disaccharide components aremade up of D-glucuronic acid and D-xylopyranose, (see "TheCarbohydrates" by W. Pigman, pages 668-669--R. L. Whistler, W. M.Corbett), the pectins and acidic polysaccharides obtainable from thesame, that is, galacturonan, acaidic polysaccharides obtainable fromplant gum (exudates), such as arabic gum, tragacanth, and finally acidicpolysaccharides derived from seaweed, such as agar and carrageenans. Asstarting material it is of course possible to use also the molecularfractions obtained by degradation of all of the above mentionedpolysacchaarides.

As organic solvents it is preferable to use aprotic solvents, such asdialkylsulphoxides, dialkylcarboxamides, such as in particular loweralkyl dialkylsulphoxides, especially dimethylsulphoxide, and lower alkyldialkylamides of lower aliphatic acids, such as dimethyl ordiethylformamide or dimethyl or diethylacetamide.

Other solvents however are to be considered which are not alwaysaprotic, such as alcohols, ethers, ketones, esters, especially aliphaticor heterocyclic alcohols and ketones with a lower boiling point, such ashexafluoro isopropanol, trifluoroethanol, and N-methylpyrrolidone.

The reaction is effected preferably at a temperature range of betweenabout 0° C. and 100° C., especially between about 25° C. and 75° C., forexample at about 30° C.

The esterification is carried out preferably by adding by degrees theesterifying agent to the above mentioned ammonium salt to one of theabove mentioned solvents, for example to dimethylsulphoxide.

As an alkylating agent it is possible to use those mentioned above,especially the hydrocarbyl halogens, for example alkyl halogens. Asstarting quaternary ammonium salts it is preferable to use the lowerammonium tetraalkylates, with alkyl groups preferably between 1 and 6carbon atoms. Mostly, hyaluronate of tetrabutylammonium is used. It ispossible to prepare these quaternary ammonium salts by reacting ametallic salt of acidic polysaccharide, preferably one of thosementioned above, especially sodium or potassium salt, in aqueoussolution with a salified sulphonic resin with a quaternary ammoniumbase.

The tetraalkylammonium salt of the acidic polysaccharide can be obtainedby freeze drying the eluate. The tetraalkylammonium salts of acidicpolysaccharides used as starting compounds of the new procedure andderiving from inferior alkyls, especially alkyls with between 1 and 6carbon atoms, are new and form another object of the present invention.Surprisingly, such salts have provided to be soluble in the abovementioned organic solvents, and for this reason the esterification ofacidic polysaccharide according to the above mentioned procedure B isparticularly easy and gives generous yields. It is therefore only byusing this kind of procedure that one can exactly dose the number ofcarboxylic groups of acidic polysaccharide which are to be esterified.

The new procedure B is very suitable especially for the preparation ofhyaluronic esters according to the present invention. In particular,therefore, as starting compounds of the new procedure, the quaternaryammonium salts of hyaluronic acid, especially those deriving from loweralkyls, and especially from alkyls with between 1 and 6 carbon atoms,are new and form a particular object of the present invention.

One variation of the previously described procedure consists in reactingpotassium salt or acidic polysaccharide sodium, suspended in a suitablesolution such as dimethylsulphoxide, with a suitable alkylating agent inthe presence of catalytic quantities of a quaternary ammonium salt, suchas iodide of tetrabutylammonium.

For the preparation of the new esters according to the present inventionit is possible to use hyaluronic acids of any origin, such as forexample the acids extracted from the above mentioned natural startingmaterials, for example from cocks' combs. The preparation of such acidsis described in literature: preferably, purified hyaluronic acids areused. According to the invention, especially used are hyaluronic acidscomprising molecular fractions of the integral acids obtained directlyby extraction of the organic materials with molecular weights varyingwithin a wide range, for example from about 90%-80% (MW=11.7-10.4million) to 0.2% (MW 30,000) of the molecular weight of the integralacid having a molecular weight of 13 million, preferably between 5% and0.2%. Such fractions may be obtained with various procedures describedin literature, such as by hydrolyzing, oxydizing, enzymatic or physicalprocedures, such as mechanical or radiational procedures. Primordialextracts are therefore often formed during these same purificationprocedures (for example see the article by Balazs et al. quoted above in"Cosmetics & Toiletries"). The separation and purification of themolecular fractions obtained are brought about by known techniques, forexample by molecular filtration.

One fraction of purified HY suitable for use according to the inventionis for example that known as "non-inflammatory-NIF-NaHA sodiumhyaluronate described by Balazs in the booklet "Healon"--A guide to itsuse in Ophthalmic Surgery D. Miller & R. Stegmann, eds. John Wiley &Sons N.Y. 81983: p. 5.

Particularly important as starting materials for the esters of thepresent invention are two purified fractions obtainable from hyaluronicacid, for example the ones extracted from cocks' combs, known as"Hyalastine" and "Hyalectin". The fraction Hyalastine has an averagemolecular weight of about 50,000 to 100,000 while the fraction Hyalectinhas an average molecular weight of between about 500,000 and 730,000. Acombined fraction of these two fractions has also been isolated andcharacterized as having an average molecular weight of about 250,000 toabout 350,000. This combined fraction may be obtained with a yield of80% of total hyaluronic acid available in the particular startingmaterial, while the fraction Hyalectin may be obtained with a yield of30% and the fraction Hyalastine with a yield of 50% of the starting HY.The preparation of these fractions is described in the Examples A-C.

The salification of HY with the above metals, for the preparation ofstarting salts for the particular esterification procedure of thepresent invention described above, is performed in a per se knownmanner, for example by reacting HY with the calculated base quantity,for example with alkaline hydrates or with basic salts of such metals,such as carbonates or bicarbonates.

In the partial esters of the present invention it is possible to salifyall the remaining carboxylic groups or only part of them, dosing thebase quantities so as to obtain the desired stoichiometric degree ofsalification. With the correct degree of salification it is possible toobtain esters with a wide range of different dissocation constants andwhich therefore give the desired pH, in solution or "in situ" at thetime of therapeutic application.

Of the new products of the present invention, of particular importancaeare the esters and their salts described above and those described inthe following illustrative Examples.

The present invention also includes modifications of the preparationprocedures of the new esters and their salts, in which a procedure isinterrupted at any given stage or started with an intermediate compoundon which the remaining stages are carried out, or in which the startingproducts are formed in situ.

The invention is illustrated by the following Examples which are notintended to be limitive of the invention.

PREPARATION EXAMPLES

The following Examples A-C describe the procedures for preparing thepreferred hyaluronic acids fractions utilized in the present invention.

EXAMPLE A Method for obtaining a mixture of Hyalastine and Hyalectinfractions having no inflammatory activity

Fresh or frozen cocks' combs, (3000 g) are minced in a meat mincer andthen carefully homogenized in a mechanical homogenizer. The paste thusobtained is placed in a stainless steel container AISI 316 or in glassand treated with 10 volumes of anhydrous acetone. The whole is agitatedfor 6 hours at a speed of 50 rpm. It is left to separate for 12 hoursand the acetone is discarded by syphoning. The acetone extraction isrepeated until the discarded acetone has reached the correct degree ofhumidity (Karl-Fischer method). The whole is then centrifuged and vacuumdried at a suitable temperature for 5-8 hours. In this way about 500-600g of dry powdered cocks' combs are obtained.

300 gr. of dry powder are exposed to enzymatic digestion with papain(0.2 g) in aqueous conditions, buffered with phosphate buffer in thepresence of a suitable quantity of hydrochloride cysteine. The resultantis agitated for 24 hours at 60 rpm keeping the temperature constant at60°-65° C. It is then cooled at 25° C. and Celite® (60 gr) is addedmaintaining the agitation for another hour. The resulting mixture isfiltered until a clear liquid is obtained. The clear liquid thenundergoes molecular ultrafiltration using membranes with a molecularexclusion limit of 30,000 in order to retain on the membrane thosemolecules with a molecular weight greater than 30,000.

The product is ultrafiltered from 5 to 6 original volumes addingdistilled water continually to the product in ultrafiltration. Theaddition of water is suspended and the ultrafiltration is continueduntil the volume is reduced to 1/3 of the original volume.

The residue liquid is rendered 0.1M by the addition of sodium chlorideand the temperature is brought to 50° C. Under agitation at 60 rpm, 45 gof cetylpyridinium chloride are added. It is agitated for 60 minutes andthen 50 g of Celite® are added. Under agitation, the temperature of thewhole is brought to 25° C. and the precipitate formed by centrifugationis gathered. The precipitate obtained is suspended in a 0.01M solutionin sodium chloride (5 liters) containing 0.05% of cetylpiridiniumchloride. The resulting suspension is agitated for 60 minutes at 50° C.;the temperature is then brought to 25° C. and the precipitate iscentrifuged. The washing operation is repeated 3 times after which theprecipitate is gathered in a receptacle containing 3 liters of a 0.05Msolution of sodium chloride containing 0.05% of cetylpyridiniumchloride. It is agitated at 60 rpm for 60 minutes and the temperature iskept constant at 25° C. for two hours. The supernatant is eliminated bycentrifugation. The procedure is repeated several times with solutionsof 0.1M sodium chloride containing 0.05% of cetylpyridinium chloride.The mixture is centrifuged and the supernatant is discarded. Theprecipitate is dispersed in a solution of 0.30M sodium chloridecontaining 0.05% of cetylpyridinium chloride (3 liters). The mixture isagitated and both the precipitate and the clear liquid are gathered.Extraction is repeated three more times on the precipitate, each timeusing 0.5 liter of the same aqueous solution.

Finally the precipitate residue is eliminated and the clear liquids areall placed together in a single container. The temperature of the liquidis brought to 50° C. under constant agitation. The liquid is thenbrought to 0.23M with sodium chloride. 1 gr of cetylpyridinium chlorideis added, and it is maintained in agitation for 12 hours.

The mixture is cooled at 25° C. and then filtered first on Celite® packand then through a filter. It then undergoes molecular ultrafiltrationagain, on a membrane with a molecular exclusion limit of 30,000ultrafiltering three initial volumes with the addition of a solution of0.33M sodium chloride. The addition of sodium chloride solution isinterrupted and the volume is reduced to 1/4 of the initial volume. Thesolution thus concentrated is precipitated under agitation (60 rpm) at25° C. with 3 volumes of ethanol (95%). The precipitate is gathered bycentrifugation and the supernatant is discarded. The precipitate isdissolved in 1 lt of 0.01M solution in sodium chloride and theprecipitation is repeated with 3 volumes of ethanol 95%.

The precipitate is gathered and washed first with 75% ethanol (3 times),then with absolute ethanol (3 times), and lastly with absolute acetone(3 times).

The product thus obtained (HYALASTINE+HYALECTIN fractions) has anaverage molecular weight of between 250,000 and 350,000.

The yield of HY is 0.6% of the original fresh tissue.

EXAMPLE B Method for obtaining the fraction Hyalastine from the mixtureobtained by the method described in Example A

The mixture obtained by the method described in Example A is dissolvedin twice distilled apyrogenetic water at the rate of 10 mg of product toeach 1 ml of water. The solution obtained is exposed to molecularfiltration through filter membranes with a molecular exclusion limit of200,000, following a concentration technique on the membrane without theaddition of water. During the ultrafiltration process through membraneswith a molecular exclusion limit of 200,000, the molecules with amolecular weight of more than 200,000 do not pass through, while thesmaller molecules pass through the membrane together with the water.During the filtration procedure no water is added, so that the volumedecreases, and there is therefore an increase in the concentration ofmolecules with a molecular weight of more than 200,000. The product isultrafiltered until the volume on top of the membrane is reduced to 10%of the initial volume. Two volumes of apyrogenetic twice distilled waterare added and it is then ultrafiltered again until the volume is reducedto 1/3. The operation is repeated twice more. The solution passedthrough the membrane is brought to 0.1M with sodium chloride and thenprecipitated with 4 volumes of ethanol at 95%. The precipitate is washed3 times with ethanol at 75% and then vacuum dried.

The product thus obtained (HYALASTINE fraction) has an average molecularweight of between 50,000 and 100,000. The yield of HY is equal to 0.4%of the original fresh tissue.

EXAMPLE C Method of obtaining the fraction Hyalectin

The concentrated solution gathered in the container on top of theultrafiltration membrane with a molecular exclusion of 200,000 as inExample B, is diluted with water until a solution containing 5 mg/ml ofhyaluronic acid is obtained, as determined by quantitative analysisbased on the dosage of glucuronic acid.

The solution is brought to 0.1M in sodium chloride and then precipitatedwith 4 volumes of ethanol at 95%. The precipitate is washed 3 times withethanol at 75% and then vacuum dried.

The product thus obtained (HYALECTIN fraction) has an average molecularweight of between 500,000 and 730,000. This corresponds to a specificfraction of hyaluronic acid with a defined length of molecular chain ofabout 2,500 to 3,500 saccharide units with a high degree of purity. Theyield of HY is equal to 0.2% of the original fresh tissue.

EXAMPLE D Preparation of the salt of tetrabutylammonium of hyaluronicacid (HY)

4.02 g of HY sodium salt (10 m.Eq.) are solubilized in 400 ml ofdistilled H₂ O. The solution is then eluted in a thermostatic column at4° C. containing 15 ml of sulphonic resin (Dowex 50×8) inTetrabutylammonium form. The eluate, free from sodium, is instantlyfrozen and freeze-dried. Yield: 6.18 g.

EXAMPLE 1 Preparation of the (partial) propyl ester of hyaluronic acid(HY)

50% of the esterified carboxylic groups

50% of the salified carboxylic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight 170,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25° C., 1.8 g (10.6 m.Eq.) of propyl iodide areadded and the resulting solution is kept at a temperature of 30° for 12hours.

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30°.

The product is then dissolved in 550 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water (5:1) and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 7.9 g of thepartial propyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 2 Preparation of the (partial) isopropyl ester of hyaluronicacid (HY)

50% of esterified carobyxlic groups

50% of salified carboyxlic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 160,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethysulfoxide at 25°, 1.8 g (10.6 m.Eq.) of isopropyl iodide areadded and the resulting solution is kept for 12 hours at 3020 .

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30°.

The produce is then dissolved in 500 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 7.8 g of thepartial isopropyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P.C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 3 Preparation of the (partial) ethyl ester of hyaluronic acid(HY)

75% of esterified carboxylic groups

25% of salified carobyxlic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 250,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethysulfoxide at 25°, 2.5 g (15.9 m.Eq.) of ethyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30°.

The produce is then dissolved in 500 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 7.9 g of thepartial ethyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 4 Preparation of the (partial) methyl ester of hyaluronic acid(HY)

75% of esterified carboxylic groups

25% of salified carboxylic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 80,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 2.26 g (15.9 m.Eq.) of methyl iodide areadded and the resulting solution is kept for 12 hours at 30°.

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30°.

The product is then dissolved in 550 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 7.8 g of thepartial methyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33. 1028-1030, (1961)].

EXAMPLE 5 Preparation of the methyl ester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 120,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 3 g (21.2 m.Eq.) of methyl iodide are addedand the solution is kept for 12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty four hours at 30°.

8 g of the ethyl ester product in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 6 Preparation of the ethyl ester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 85,000corresponding to 20 m.Eq. of a monomeric unit are solubized in 620 ml ofdimethylsulfoxide at 25°, 3.3 g (21.2 m.Eq.) of ethyl iodide are addedand the solution is kept for 12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty-four hours at 30°.

8 g of the ethyl ester product in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 7 Preparation of the propyl ester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 3.6 g (21.2 m.Eq.) of propyl iodide areadded and the solution is kept for 12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty-four hours at 30°.

8.3 g of the propyl ester product in the title are obtained.Quantitative determination of the ester groups is carried out using themethod of R. H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030,(1961)].

EXAMPLE 8 Preparation of the (partial) butyl ester of hyaluronic acid(HY)

50% of esterified carboxylic groups

50% of salified carboxylic groups (Na).

12.4 g of HY tetrabutylammonium salt with a molecular weight of 620,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 1.95 g (10.6 m.Eq.) of n-butyl iodide areadded and the resulting solution is kept for 12 hours at 30°.

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30°.

The product is then dissolved in 550 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 8 g of thepartial butyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 9 Preparation of the (partial) ethoxycarbonylmethyl ester orhyaluronic acid (HY)

75% of esterified carobyxlic groups

25% of salified carboxylic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 180,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 2 g of tetrabutylammonium iodide and 1.84 g(15 m.Eq.) of ethyl chloroacetate are added and the resulting solutionof kept for 24 hours at 30°.

A solution containing 62 ml of water and 9 g of sodium chloride is addedand the resulting mixture is slowly poured into 3,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed three times with 500 ml of acetone/water 5:1 and three times withacetone and finally vacuum dried for eight hours at 30° C.

The product is then dissolved in 550 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml di acetone and finally vacuum dried for 24 hours at 30°. 10 g of thepartial ethoxycarbonyl methyl ester compound in the title are obtained.Quantitative determination of the ethoxylic ester groups is carried outusing the method of R. H. Cundiff and P. C. Markunas [Anal. Chem. 33,1028-1030, (1961)].

EXAMPLE 10 Preparation of the (partial) cortisone ester (C₂₁) ofhyaluronic acid (HY)

20% of esterified carboxylic groups

80% of salified carboxylic groups (Na)

6.2 g of HY tetrabutylammonium salt with a molecular weight of 105,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.850 g (2 m.Eq.) of 21-bromo-4-pregnene-17α-ol-3, 11, 20-trione are added and the resulting solution is kept for24 hours at 30°.

A solution containing 100 ml of water and 5 g of sodium chloride isadded and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which filteredand washed three times with 100 ml of acetone/water 5:1 and three timeswith acetone and finally vacuum dried for eight hours at 30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.5 g of thepartial cortisone ester compound in the title are obtained. Quantitativedetermination of cortisone, after mild alkaline hydrolysis with ahydroalcoholic solution of Na₂ CO₃ and extraction with chloroform, iscarried out according to British Pharmacopea, 1980, p. 127.

EXAMPLE 11 Preparation of the (partial) hydrocortisone ester (C₂₁) ofhyaluronic acid (HY)

20% of esterified carboxylic groups

80% of salified carboxylic groups (Na).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 80,000corresponding to 20 m.Eq. of a monomeric unit are solublized in 310 mlof dimethylsulfoxide at 25°, 0.850 g (2 m.Eq.) of21-bromo-4-pregnene-11β, 17 α-diol-3,20-dione are added and theresulting solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.4 g of thepartial hydrocortisone ester compound in the title are obtained.Quantitative determination of hydrocortisone, after mild alkalinehydrolysis with hydroalcoholic solution of Na₂ CO₃ and extraction withchloroform, is carried out according to British Pharmacopea, 1980, p.224.

EXAMPLE 12 Preparation of the (partial) fluorocortisone ester (C₂₁) ofhyaluronic acid (HY)

20% of esterified carboxylic groups

80% of salified carboxylic groups (Na)

6.2 g of HY tetrabutylammonium salt with a molecular weight of 80,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.89 g (2 m.Eq.) of9-fluoro-21-bromo-4-pregnene-11β, 17 α-diol-3,20-dione are added and theresulting solution is kept for 12 hours at 30°.

A solution is then added containing 62 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.6 g of thepartial fluorocortisone compound in the title are obtained. Quantitativedetermination of fluorocortisone, after mild alkaline hydrolysis withhydroalcoholic solution of Na₂ CO₃ and extraction with chloroform, iscarried out according to British Pharmacopea, 1980, p. 196.

EXAMPLE 13 Preparation of the (partial) desoxycorticosterone ester (C₂₁)of hyaluronic acid (HY)

20% of esterified carboxylic groups

80% of salified carboxylic groups (Na)

6.21 g of HY tetrabutylammonium salt with a molecular wieght of 105,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.661 g (2 m.Eq.) of 21-bromo-4-pregnene-3,20-dione are added and the resulting solution is kept for 24 hours at30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 300 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 1,500 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 100 ml of acetone/water 5:1 and three times with 100ml of acetone and finally vacuum dried for 24 hours at 30°. 4.5 g of thepartial desoxycorticosterone ester compound in the title are obtained.Quantitative determination of desoxycorticosterone, after mild alkalinehydrolysis with hydroalcoholic solution of Na₂ CO₃ and extraction withchloroform, is carried out according to British Pharmacopea, 1980, p.137.

EXAMPLE 14 Preparation of the (mixed ethanol and cortisone ester (C₂₁)of hyaluronic acid (HY)

80% of the carboxylic groups esterified with ethanol

20% of the carboxvlic groups esterified with cortisone (C₂₁).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq. of a monomeric unit are solublized in 310 mlof dimethylsulfoxide at 25°, 1.25 (8 m.Eq.) of ethyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-17 α-ol-3, 11, 20-trione areadded and the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly pourd into 2,000 ml ofacetone under constant agitation. A precipitate is formed which is thenfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the mixed ethanol and cortisone ester compound in the title areobtained. Quantitative determination of cortisone, after mild alkalinehydrolysis is with hydroalcoholic solution of Na₂ CO₃ and extractionwith chloroform, is carried out according to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundif and P. C. Markunas Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 15 Preparation of the (mixed) ethanol and hydrocortisone ester(C₂₁) of hyaluronic acid (HY)

80% of carboxvlic groups esterified with ethanol

20% of carboxylic groups esterified with hydrocortisone (LC₂₁)

6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 1.25 g M.Eq.) of ethyl iodide are added andthe solution is kept at 30° for 12 hours.

0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-11β, 17 α-diol-3,20-dione areadded and the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the mixed ethanol and hydrocortisone ester compound in thetitle are obtained. Quantitative determination of hydrocortisone, aftermild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ andextraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 16 Preparation of the (mixed) ethanol and fluorocortisone ester(C₂₁) of hyaluronic acid (HY)

80% of carboxylic groups eserified with ethanol

20% of carboxylic groups esterified with fluorocortisone (C₂₁).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 1.25 g (8 m.Eq.) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.89 g (2 m.Eq.) of 9 α-fluoro-21-bromo-4-pregnene-11 β, 17α-diol-3,20-dione are added and the solution is kept for 24 hours at30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the mixed ethanol and fluorocortisone ester compound featuredin the title are obtained. Quantitative determination offluorocortisone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 17 Preparation of the (mixed) ethanol and desoxycorticosteroneester (C₂₁) of hyaluronic acid (HY)

80% of carboxylic groups esterified with ethanol

20% of carboxylic groups esterified with desoxycorticosterone (C₂₁)

6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 1.25 g (8 m.Eq.) of ethyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

0.661 g (2 m.Eq.) of 21-bromo-4-pregnene-3, 20-dione are added and thesolution of kept for 24 hours at 30°. A solution is then addedcontaining 100 ml of water and 5 g of sodium chloride and the resultingmixture is slowly poured into 2,000 ml of acetone under constantagitation. A precipitate is formed which is filtered and washed threetimes with 100 ml of acetone/water 5:1 and three times with 100 ml ofacetone and finally vacuum dried for eight hours at 30°.

4.6 g of the mixed ethanol and desoxycorticosterone ester compound inthe title are obtained. Quantitative determination ofdesoxycorticosterone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas [Anal Chem. 33, 1028-1030 (1961)].

EXAMPLE 18 Preparation of the (partial and mixed) ethanol anddesoxycorticosterone ester of hyaluronic acid (HY)

40% of carboxylic groups esterified with desoxycorticosterone (C₂₁)

40% of salified carboxylic qroups (Na).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.62 g (4 m.Eq.) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-3, 20-dione are added and thesolution is kept for 24 hours at 30°. A solution is then addedcontaining 100 ml of water and 5 g of sodium chloride and the resultingmixture is slowly poured into 2,000 ml of acetone under constantagitation. A precipitate is formed which is filtered and washed threetimes with 100 ml of acetone/water 5:1 and three times with 100 ml ofacetone and finally vacuum dried for eight hours at 30°.

4.5 g of the partial and mixed ethanol and desoxycorticosterone estercompound in the title are obtained. Quantitative determinations ofdesoxycorticosterone, after mild alkaline hydrolysis with hydroalcoholicsolution of Na₂ CO₃ and extraction with chloroform, is carried outaccording to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 19 Preparation of the (partial and mixed) ethanol and cortisoneester of hyaluronic acid (HY)

40% of carboxylic groups esterified with ethanol

20% of carboxylic groups esterified with cortisone (C₂₁)

40% of salified carboxylic groups (Na).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 125,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.62 g (4 m.Eq.) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq.) of 21-bromo-4-pregnen-17 α-ol-3, 11,20-trione are addedand the solution is kept for 24 hours at 30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.5 g of the partial and mixed ethanol and cortisone compound in thetitle are obtained. Quantitative determination of cortisone, after mildalkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ extractionwith chloroform, is carried out according to British Pharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 20 Preparation of the (partial and mixed) ethanol andhydrocortisone ester (LC₂₁) of hyaluronic acid (HY)

40% of carboxylic groups esterified with ethanol

20% of carboxylic groups esterified with hydrocortisone (C₂₁)

40% of salified carboxylic groups (Na).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 70,000corresponding to 10 m.Eq. of a monomeric unit are solubilized in 310 mlof dimethylsulfoxide at 25°, 0.62 g (4 m.Eq.) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.85 g (2 m.Eq.) of 21-bromo-4-pregnene-11 β, 17 α-diol-3,20-dione areadded and the solution is kept for 24 hours at 30°.

A solution is then added containing 200 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml of acetone and finally vacuum dried for eighthours at 30°.

4.5 g of the partial and mixed ethanol and hydrocortisone ester compoundin the title are obtained. Quantitative determination of hydrocortisone,after mild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃and extraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 21 Preparation of the (partial and mixed) ethanol andfluorocortisone esters (C₂₁) of hyaluronic acid (HY)

40% of carboxylic groups esterified with ethanol

20% of carboxylic groups esterified with fluorocortisone (C₂₁)

40% of salified carboxylic groups (Na).

6.2 g of HY tetrabutylammonium salt with a molecular weight of 65,000corresponding to 20 m.Eq. of a monomeric unit are solublized in 310 mlof dimethylsulfoxide at 25°, 0.62 g (4 m.Eq.) of ethyl iodide are addedand the solution is kept for 24 hours at 30°.

0.89 g (2 m.Eq.) of 9 α-fluoro-21-bromo-4-pregnene-11β, 17α-diol-3,20-dione are added and the solution is kept for 24 hours at30°.

A solution is then added containing 100 ml of water and 5 g of sodiumchloride and the resulting mixture is slowly poured into 2,000 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 100 ml of acetone/water 5:1 andthree times with 100 ml di acetone and finally vacuum dried for eighthours at 30°.

4.6 g of the partial and mixed ethanol and fluorocortisone ester in thetitle are obtained. Quantitative determination of fluorocortisone, aftermild alkaline hydrolysis with hydroalcoholic solution of Na₂ CO₃ andextraction with chloroform, is carried out according to BritishPharmacopea, 1980.

Quantitative determination of the ethoxyls is carried out according toR. H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030 (1961)].

EXAMPLE 22 Preparation of the n-pentyl ester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 620,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 3.8 g (25 m.Eq.) of n-pentyl bromide and0.2 g of iodide tetrabutylammonium are added, the solution is kept for12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty four hours at 30°.

8.7 g of the n-pentyl ester product in the title are obtained.Quantitative determination of the ester groups is carried out using themethod described on pages 169-172 of Siggia S. and Hann J. G."Quantitative organic analysis via functional groups" 4th edition JohnWiley and Sons.

EXAMPLE 23 Preparation of the isopentyl ester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethysulfoxide at 25°, 3.8 g (25 m.Eq.) of isopentyl bromide and0.2 g of tetrabutylammonium iodide are added, the solution is kept for12 hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty four hours at 30°.

8.6 g of the isopentyl ester product featured in the title are obtained.Quantitative determination of the ester groups is carried out accordingto the method described on pages 169-172 of Siggia S. and Hanna J. G."Quantitative organic analysis via functional groups" 4th edition, JohnWiley and Sons.

EXAMPLE 24 Preparation of the benzylester of hyaluronic acid (HY)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 170,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 4.5 g (25 m.Eq.) of benzyl bromide and 0.2g of tetrabutylammonium iodide are added, the solution is kept for 12hours at 30°.

The resulting mixture is slowly poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is formed which is filtered andwashed four times with 500 ml of ethyl acetate and finally vacuum driedfor twenty four hours at 30°.

9 g of the benzyl ester product in the title are obtained. Quantitativedetermination of the ester groups is carried out according to the methoddescribed on pages 169-172 of Siggia S. and Hanna J. G. "Quantitativeorganic analysis via functional groups" 4th edition, John Wiley andSons.

EXAMPLE 25 Preparation of the β-phenylethyl ester of hyaluronic acid(HY).

12.4 g of HY tetrabutylammonium salt with a molecular weight of 125,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethylsulfoxide at 25°, 4.6 g (25 m.Eq.) of 2-bromoethylbenzene and185 mg of tetrabutylammonium iodide are added, and the solution is keptfor 12 hours at 30°.

The resulting mixture is slowly. poured into 3,500 ml of ethyl acetateunder constant agitation. A precipitate is thus formed which is thenfiltered and washed four times with 500 ml of ethyl acetate and finallyvacuum dried for twenty four hours at 30°.

9.1 g of the β-phenylethyl ester in the title are obtained. Quantitativedetermination of the ester groups is carried out according to the methoddescribed on page 168-172 of Siggia S. and hanna J. G. "Quantitativeorganic analysis via functional groups" 4th edition, John Wiley andSons.

EXAMPLE 26 Preparation of the benzyl ester of hyaluronic acid (HY)

3 g of the potassium salt of HY with a molecular weight of 162,000 aresuspended in 200 ml of dimethylsulfoxide; 120 mg of tetrabutylammoniumiodide and 2.4 g of benzyl bromide are added.

The suspension is kept in agitation for 48 hours at 30°. The resultingmixture is slowly poured into 1,000 ml of ethyl acetate under constantagitation. A precipitate is formed which is filtered and washed fourtimes with 150 ml of ethyl acetate and finally vacuum dried for twentyfour hours at 30°.

3.1 g of the benzyl ester product in the title are obtained.Quantitative determination of the ester groups is carried out accordingto the method described on pages 169-172 of Siggia S. and Hanna J. G."Quantitative organic analysis via functional groups" 4th edition, JohnWiley and Sons.

EXAMPLE 27 Preparation of streptomycine salt of hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic groups salified with streptomycine

243 mg of streptomycine sulphate (1 m.Eq.) are solubilized in 20 ml ofwater. The solution is eluted in a thermostatic column at 5° containing2 ml of quaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HY and 25% sodium salt (corresponding to 1m.Eq. of a monomeric unit relative to the non-esterified carboxyl), aresolubilized in 400 ml of water. The solution is eluted in a thermostaticcolumn at 20° and containing 2 ml of sulphonic resin (Dowex 50×8) in H⁺form.

The sodium-free eluate is gathered under agitation in the solution ofstreptomycine base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.7 g.

Microbiological determination on B.subtilis ATCC 6633 in comparison withstreptomycine standard, shows a content of 10.9% in weight ofstreptomycine base, corresponding to the theoretically calculatedcontent.

EXAMPLE 28 Preparation of the erythromycin salt of hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic groups salified with erythromycin

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto m.Eq. of a monomeric unit relative to the non-esterified carboxyl),are solublized in 400 ml of water. The solution is eluted in athermostatic column at 20° containing 2 ml of sulfonic resin (Dowex 50×8) in H⁺ form.

To the sodium-free eluate are added 734 mg of erythromycin base (1m.Eq.). The resulting solution is instantly frozen and freeze-dried.Yield: 2.1 g.

Microbiological determination on St. aureus ATCC 6538 in comparison tostandard erythromycin, shows a content of 31.7% in weight oferythromycin base, corresponding to the theoretically calculated weight.

EXAMPLE 29 Preparation of the neomycin salt of a hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic groups salified with neomycin

152 mg of neomycin sulfate (1 m.Eq.) are solubilized in 20 ml of water.The solution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HY and sodium salt al 25% (correspondingto 1 m.Eq. of monomeric unit relative to the non-esterified carboxyl),are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofneomycin base The resulting solution is instantly frozen andfreeze-dried. Yield: 1.65 g.

Microbiological determination carried out on St. aureus ATCC 6538 incomparison to standard neomycin, shows a content of 6.1% in weight ofneomycin base, corresponding to the theoretically calculated value.

EXAMPLE 30 Preparation of the gentamicin salt of hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic groups salified with gentamicin

145 mg of gentamicin sulfate are solubilized in 10 ml of water. Thesolution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowec 1×8) in OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl),are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofgentamicin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.7 g.

Microbiological determination carried out on S. epidermidus ATCC 12228in comparison to standard gentamicin, shows a content of 6.50% in weightof gentamicin base, corresponding to the theoretically calculated value.

EXAMPLE 31 Preparation of the amikacin salt of hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic Groups salified with amikacin 147 mg of amikacin base(1 m.Eq.) are solubilized in 20 ml of water.

147 mg of amikacin (1 m.Eq.) are solubilized in 20 ml of water.

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl),are solubilized in 400 ml of wter. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofamikacin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.70 g.

Microbiological determination carried out on St. aureus ATCC 29737 incomparison to standard amikacin, shows a content of 8.5% in weight ofamikacin base, corresponding to the theoretically calculated value.

EXAMPLE 32 Preparation of the kanamycin salt of hyaluronic acid (HY)partially estified with ethanol

75% of carboxylic qroups esterified with ethanol

25% of carboxylic qroups salified with kanamycin

146 mg of kanamycin sulfate (1 m.Eq.) are solubilized in 20 ml of water.The solution is eluted in a thermostatic column at 5° containing 2 ml ofquaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The sulphate-free eluate is gathered in a thermostatic container at atemperature of 5°.

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl),are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofkanamycin base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1.5 g.

Microbiological determination carried out on B. subtilis ATCC 6633 incomparison to standard kanamycin, shows a content of 7% in weight ofkanamycin base, corresponding to the theoretically calculated value.

EXAMPLE 33 Preparation of the pilocarpine salt of hyaluronic acid (HY)partially esterified with ethanol

75% of carboxylic groups esterified with ethanol

25% of carboxylic groups salified with pilocarpine

245 mg of pilocarpine hydrochloride (1 m.Eq.) are solubilized in 20 mlof water. The solution is eluted in a thermostatic column at 5°containing 2 ml of quaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The chloride-free eluate is gathered in a thermostatic container at 5°.

1.6 g of a 75% ethyl ester of HY and sodium salt at 25% (correspondingto 1 m.Eq. of a monomeric unit relative to the non-esterified carboxyl),are solubilized in 400 ml of water. The solution is eluted in athermostatic column at 20° and containing 2 ml of sulfonic resin (Dowex50×8) in H⁺ form.

The sodium-free eluate is gathered under agitation in the solution ofpilocarpine base. The resulting solution is instantly frozen andfreeze-dried. Yield: 1/89 g.

EXAMPLE 34 Preparation of the (partial propyl) ester of hyaluronic acid(HY)

85% of esterified carboxylic groups

15% of salified carboxylic groups (Na)

12.4 g of HY tetrabutylammonium salt with a molecular weight of 165,000corresponding to 20 m.Eq. of a monomeric unit are solubilized in 620 mlof dimethysulfoxide at 25°, 2.9 g (17 m.Eq.) of propyl iodide are addedand the resulting solution is kept for 12 hours at 30°.

A solution is then added containing 62 ml of water and 9 g of sodiumchloride and the resulting mixture is slowluy poured into 3,500 ml ofacetone under constant agitation. A precipitate is formed which isfiltered and washed three times with 500 ml of acetone/water 5:1 andthree times with acetone and finally vacuum dried for eight hours at30°.

The product is then dissolved in 550 ml of water containing 1% of sodiumchloride and the solution is slowly poured into 3,000 ml of acetoneunder constant agitation. A precipitate is formed which is filtered andwashed twice with 500 ml of acetone/water 5:1 and three times with 500ml of acetone and finally vacuum dried for 24 hours at 30°. 8 g of thepartial propyl ester compound in the title are obtained. Quantitativedetermination of the ester groups is carried out using the method of R.H. Cundiff and P. C. Markunas [Anal. Chem. 33, 1028-1030, (1961)].

EXAMPLE 35 Preparation of the pilocarpine salt of hyaluronic acid (HY)partially esterified with n-propanol

85% of carboxylic groups esterified with n-propanol

15% of carboxylic groups salified with pilocarpine

245 mg of pilocarpine hydrochloride (1 m.Eq.) are solubilized in 10 mlof water. The solution is eluted in a thermostatic column at 5°containing 2 ml of quaternary ammonium resin (Dowex 1×8) in OH⁻ form.

The chloride-free eluate is gathered in a thermostatic container at 5°.

4.1 g. o f the p ropyl ester of HY 85 % and tetrabutylammonium salt at15% (corresponding to 1 m.Eq. of a monomeric unit relative to thenon-esterified carboxyl) are solubilized in 100 ml of dimethylsulfoxide.The solution is eluted in a thermostatic column at 20° containing 2 mlof damp sulfonic resin (Dowex 50×8) in H⁺ form.

The eluate is gathered under agitation in the solution of pilocarpinebase. The resulting solution is precipitated with ethyl acetate (600ml).

The precipitate is filtered and washed four times with 200 ml of ethylacetate and finally vacuum dried for 24 hours at 30°. 3.5 g of thecompound featured in the title are obtained.

EXAMPLE 36 Preparation of the ethyl ester of an acidic polysaccharideproducted by Rhinocladiella eliator

The acidic polysaccharide produced by Rhinocladiella eliator MangenotNRRL YB-4613 is used (P. R. Watson, P. A. Sandford, K. A. Burton, M. C.Cadmus and A. Jeanes - Carbohydr. Res. 46, 259-265 (1976); L. Kenne, B.Lindberg, K. Peterson and P. Unger, Carbohydr. Res. 84, 184-186 (1980).It is made up of units of 2-acetamido-2-deoxy-D-glucuronic acidconnected by bonds 1→4. ##STR1##

5.2 g of the potassium salt of this acidic polysaccharide, correspondingto 20 mEq of a monomeric unit, are suspended in 250 ml ofdimethylsulfoxide. While the mixture is kept in agitation, 200 mg oftetrabutylammonium iodide are added at 3° C. and then slowly 3.5 g ofmethyl iodide. The mixture is kept in agitation for 48 hours at 35° C.,after which it is slowly poured into 800 ml of ethyl acetate, keeping itunder constant agitation. A precipitate is formed which is filtered andwashed four times with 150 ml of ethyl acetate and lastly vacuum dried.4 g of the ethyl ester product in the title are thus obtained, in whichall the carboxylic groups are esterified. Quantitative determination ofthe ester groups is carried out by the method of R. H. Cundiff and P. C.Markanas Anal. Chem. 33, 1028-1030 (1961).

EXAMPLE 37 Preparation of the ethyl ester of acid polysaccharideproduced by Rhinocladiella eliator

10.0 g of the tetrabutylammonium salt of the acidic polysaccharide usedas starting substance in Example 36, corresponding to 20 mEq of amonomeric unit, are treated with 800 ml of dimethylsulfoxide at 30° C.3.3 g (21.2 mEq) of ethyl iodide are added and the solution is keptunder agitation for 48 hours at 30° C. The resulting mixture is slowlypoured into 4000 ml of ethyl acetate while kept under constantagitation. A precipitate is formed which is filtered and washed fourtimes with 500 ml of ethyl acetate and lastly vacuum dried.

3 g of the ethyl ester product in the title are obtain, in which all thecarboxylic groups are esterified.

Quantitative determination of the ester groups is carried out by themethod of R. H. Cundiff and P. C. Markanas [Anal. Chem. 33, 1028-1030,(1961)].

EXAMPLE 38 Preparation of the ethyl ester of the acidic polysaccharideproduced by Rhinocladiella Mansoni

Acidic polysaccharide produced by Rhinocladiella Mansoni NRRL Y-46272[(A. Jeanes, K. A. Burton, M. C. Cadmus, C. A. Knutson, G. L. Rowin andP. A. Sandford--Nature (London) 233, 259-260 (1971); P. A. Sandford, P.A. Watson and A. P. Jeanes--Carbohydr. Res. 29, 153-166 (1973)]. It ismade up of units formed by one molecule of2-acetamido-2-deoxy-D-glucuronic acid and two molecules ofN-acetyl-D-glucasamine connected by bonds 1→3. ##STR2##

18.2 g of tetrabutylammonium salt of this acidic polysaccharide,corresponding to 20 mEq of a monomeric unit, are treated with 1000 ml ofdimethylsulfoxide at 30° C. Under agitation, 3.3 g (21.2 mEq) of ethyliodide and the solution is kept at 30° C. for 24 hours, after which itis slowly poured into 4000 ml of ethyl acetate, keep it under constantagitation. A precipitate is formed which is filtered and washed fourtimes with 500 ml of ethyl acetate and lastly vacuum dried.

11 g of the product featured in the title are obtained, in which all thecarboxylic groups are esterified. Quantitative determination of theester groups is carried out according to the method of R. H. Cundiff andP. C. Markanas [Anal. Chem. 33, 1028-1030, (1961)].

BIOLOGICAL ACTIVITY STUDIES 1) Anti-inflammatory Activity Studies

The technical effect of the new esters and of the new medicamentsaccording to the invention may be demonstrated for example by placing inevidence the antiinflammatory activity of some partial esters ofhyaluronic acid with antiphlogistic corticosteroids, measured in themodel of exudative phlogosis induced by dextran in rabbit eye.

Materials

9 hyaluronic esters of cortisone, hydrocortisone and fluorocortisone(9 - fluorohydrocortisone) identified by the code names HYC1-HYC9 weretested. Table 1 describes these compounds and gives the percentages ofthe number of carboxylic groups of HY which are esterified with theabove corticosteroids, and where applicable the percentage esterifiedwith simple aliphatic alcohols and those salified with alkaline metals(Na):

The activity of the compounds of Table 1 was compared with thecorresponding cortisones.

                                      TABLE 1                                     __________________________________________________________________________             % CARBOXYLS   CORTI-   % CARBOXYLS  ALIFATIC                                                                             % CARBOXYLS                        ESTERIF. WITH COSTEROID                                                                              ESTERIF. WITH                                                                              ALCOHOL                                                                              SALIFIED                  COMPOUND CORTICOSTEROIDS                                                                             ASSAY (p/p)                                                                            ALIPHATIC ALCOHOL                                                                          ASSAY (p/p)                                                                          WITH                      __________________________________________________________________________                                                        Na                        HYC1     20 CORTISONE  15.5%    --                  80                        HYC2     20 HYDROCORTISONE                                                                           15.6%    --                  80                        HYC3     20 FLUDROCORTISONE                                                                          16.2%    --                  80                        HYC4     20 CORTISONE  15.3%    80 ETHANOL   7.84%  /                         HYC5     20 HYDROCORTISONE                                                                           15.4%    80 ETHANOL   7.83%  /                         HYC6     20 FLUDROCORTISONE                                                                          16.1%    80 ETHANOL   7.77%  /                         HYC7     20 CORTISONE  15.4%    40 ETHANOL   3.94%  40                        HYC8     20 HYDROCORTISONE                                                                           15.5%    40 ETHANOL   3.94%  40                        HYC9     20 FLUDROCORTISONE                                                                          16.1%    40 ETHANOL   3.91%  40                        __________________________________________________________________________

All the derivatives, except for HYC4, HYC5 and HYC6 (dissolved in DMSO)were dissolved in saline (2 mg/ml).

Method

Aseptic (exudative) phlogosis was induced in 48 rabbits by intraocularinjection of dextran (1% in saline, 0.1 ml). The various products wereadministered by instillation in the right eye (RE) of the rabbits, whilein the left eye (LE) only vehicle was instilled.

The treatment (3 drops every 6 hours) was begun immediately after theinjection of dextran and was continued for 16 days.

Ophthalmic Examination

Both eyes of each rabbit were observed through a slit lamp. Inparticular the following were examined: the state of the conjunctiva andcorneal epithelium, the anterior chamber (presence of Tyndall effect),state of the iris and of the posterior segment of the eye. With aGoldmann lens, the state of the back of the eye was examined. Thepresence of signs of inflammation (hyperemia, exudate, cloudiness of theliquids, etc.) was recorded. The percentage of the eyes which did notpresent any signs of phlogisis was then calculated. Results

As can be observed from the results reported in Table 2, the HYCderivatives all proved to possess a considerable antiinflammatoryactivity consistently superior to that of the corresponding cortisonestested in parallel, reduced not only the percentage of eyes withphlogosis on each day of observation, but also reducing the duration ofinflammation. The most efficient of these derivatives seem to be HYC4,HYC5 and HYC6, presumably because they are more lipophilic.

                  TABLE 2                                                         ______________________________________                                        Antiinflammatory effect of the HYC derivatives                                (hyaluronic esters) on dextran-induced aseptic                                (exudative) phlogosis in rabbit eye                                           Days from start of phlogosis                                                  Treatment                                                                             2     4     6    8    10   12    14    16                             ______________________________________                                        Corti-  0.0   0.0   0.0  0.0  25.0 50.0  100.0 100.0                          sone (4)                                                                      Vehicle (4)                                                                           0.0   0.0   0.0  0.0  0.0  25.0  25.0  50.0                           Hydro-  0.0   0.0   0.0  25.0 25.0 50.0  100.0 100.0                          corti-                                                                        sone (4)                                                                      Vehicle (4)                                                                           0.0   0.0   0.0  0.0  0.0  25.0  50.0  100.0                          Fluoro- 0.0   0.0   0.0  0.0  25.0 50.0  100.0 100.0                          cortis. (4)                                                                   Vehicle (4)                                                                           0.0   0.0   0.0  0.0  0.0  50.0  50.0  100.0                          HYC1 (4)                                                                              0.0   0.0   0.0  25.0 50.0 50.0  100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  100.0                          HYC2 (4)                                                                              0.0   0.0   0.0  25.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  100.0                          HYC3    0.0   0.0   0.0  25.0 25.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  100.0                          HYC4 (4)                                                                              0.0   0.0   25.0 50.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  100.0                          HYC5 (4)                                                                              0.0   0.0   25.0 50.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  100.0                          HYC6 (4)                                                                              0.0   0.0   25.0 50.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  25.0  50.0                           HYC7 (4)                                                                              0.0   0.0   0.0  25.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 25.0  50.0  50.0                           HYC8 (4)                                                                              0.0   0.0   0.0  25.0 50.0 100.0 100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 50.0  50.0  50.0                           HYC9 (4)                                                                              0.0   0.0   0.0  25.0 25.0 50.0  100.0 100.0                          Vehicle (4)                                                                           0.0   0.0   0.0  0.0  00.0 50.0  50.0  100.0                          ______________________________________                                    

Values are expressed as percentages (number of eyes without signs ofphlogosis out of the total number of eyes treated). In brackets are thenumber of treated eyes.

2) Absorption and Bioavailability Studies

The technical effect of the new products according to the presentinvention may be demonstrated by a study of the absorption and of thebioavailability of some derivatives of hydrocortisone with hyaluronicacid. The derivatives used are those described above and identified asHYC2, HYC5 and HYC8.

Materials and Methods Animals

Male Sprague-Dawley rats, with a body weight of 250-350 gr were used,obtained from Charles River-Calco (Como), fed ad libitum with water andcompound feed in pellets, with the code name of 4RF 21, produced by"Italiana Mangimi", licensee of Charles River.

Treatment

Hydrocortisone was administered in the form of sodium hemisuccinate saltat the dose of 1.34 mg/kg (corresponding to 1 mg/kg of hydrocortisonebase) by general intravenous route and at the dose of 1.34 mg/kg and 2.68 mg/kg (corresponding to 2 mg/kg of hydrocortisone base) bysubcutaneouos route (the i.v. route was considered in order to determinethe pharmacokinetic parameters which serve as a comparison for theevaluation of absorption of any other administration route).

The three HYC derivatives were administered by subcutaneous route at thedose of 6.5 and 13 mg/kg (doses corresponding to about 1 and 2 mg/kg inhydrocortisone base). All the various products were dissolved in sterilesaline, except for HYC5 which, being insoluble in completely aqueoussolutions, was first solubilized with the addition of the minimumquantity necessary of dimethyulsulphoxide, and then brought to the rightvolume with saline. All the compounds were injected at a constant volumeof 1 ml/kg.

Gathering of the Plasma Samples

After administration, 0.3 ml of blood was drawn from each animal bycardiac puncture in the presence of anticoagulant (sodium heparin).

Blood drawing times were as follows: *15 mins, 30 mins, 60 mins, 120mins, 180 min, 300 mins, 360 mins, 420 mins, 480 mins (*limited to theintraveneous route).

Dosage of Hydrocortisone

The hydrocortisone was dosed by radioimmunoassay method (Cortisolo Kit,Biodata, cod. 10394) using iodate tracing. The precision and accuracy ofthe method, determined on six repeats (double) of a control serum with aknown control assay, proved to be 3.03% and 0.021% respectively. Thelinearity of the method comes between 1 and 1000 ng/ml. The observationlimit is 1 ng/ml.

The dosage of the cortisolemia in the rat is not influenced either bythe base levels or by the circadian rhythms of this hormone, as themetabolic pattern of the endogenous glucocorticoid hormones in the rat,leads to the production of corticosterone and not cortisol (see E. L.Green: Biology of the laboratory mouse.)

Preliminary proof has demonstrated that the dosage method is specificonly for free cortisol. The anticortisol antibody does not present anyform of competition towards any of the three HYC derivatives.

Results

In Table 3 are reported the results of the average plasma levels ofhydrocortisone, after i.v. and s.c. injection (1 and 2 mg/kg). It shouldbe emphasized that, after s.c. injection, there is a quite rapidabsorption of the product (Tmax evaluated at about 30 mins, Cmax thesame as the i.v. route levels at the same dose). In Table 4 are reportedthe pharmacokinetic parameters of cortisol calculated graphically fromthe plasmatic decline curves. In Table 3 are reported the average levelsof cortisol after subcutaneous administration of the three HYCderivatives at doses of 6.5 and 13 mg/kg (corresponding to about 1 and 2mg/kg in hydrocortisone base). In Table 4 are reported thepharmacokinetic parameters relative to cortisol calculated graphicallywith the method of residues from the plasma absorption curve of thethree HYC products.

It should be noted that the kinetics of hydrocortisone released by thethree derivatives with hyaluronic acid are not linear; that is, nodirect relationship exists between the dose-dependent parameters such asthe area beneath the plasma decline curve and the plasma levels. Sincethe kinetics of cortisol are themselves linear and a first rate modelresults, it can be deduced that the saturation process in the case ofthe HYC derivatives is the hydrolisis of the ester bond betweenhyaluronic acid and cortisolo. This phase (tending towards zero ratekinetics) is not itself connected with the absorption of the activeprinciple and therefore the kinetics of the three HYC's were likewiseresolved according to a first rate model.

                                      TABLE 3                                     __________________________________________________________________________    Average plasmatic levels of hydrocortisone after s.c. administration          of the derivatives HYC.sub.2 --HYC.sub.5 --HYC.sub.8 (6.5 and 13 mg/kg)       in comparison                                                                 to the corresponding doses (1-2 mg/Kg) of hydrocortisone (average of 4        values)                                                                       AVERAGE PLASMATIC LEVELS                                                      (mg/ml)                                                                       s.c. HYDROCORTISONE                                                           TIME i.v.           s.c. HYC.sub.2                                                                           s.c. HYC.sub.5                                                                           s.c. HYC.sub.8                      minutes                                                                            1 mg/kg                                                                            1 mg/kg                                                                            2 mg/kg                                                                            6.5 mg/kg                                                                           13 mg/kg                                                                           6.5 mg/kg                                                                           13 mg/kg                                                                           6.5 mg/kg                                                                           13 mg/kg                      __________________________________________________________________________     15 min                                                                            154.57                                                                    30 min                                                                            88.50                                                                              86.29                                                                              196.32                                                                             27.02 32.32                                                                              33.41 26.55                                                                              33.75 34.62                          60 min                                                                            59.62                                                                              61.27                                                                              142.12                                                                             35.67 50.51                                                                              49.52 38.77                                                                              45.23 51.69                         120 min                                                                            46.97                                                                              49.10                                                                              84.34                                                                              42.32 62.42                                                                              60.52 41.56                                                                              44.75 108.01                        180 min                                                                            39.35                                                                              23.44                                                                              50.02                                                                              37.35 58.44                                                                              61.17 44.81                                                                              41.80 76.64                         300 min                                                                            29.78                                                                              11.98                                                                              24.53                                                                              32.27 51.68                                                                              --    32.51                                                                              30.00 54.66                         360 min                                                                            24.49                                                                               9.96                                                                              20.15                                                                              26.96 48.17                                                                              42.44 31.92                                                                              26.10 47.17                         420 min                                                                            21.88                                                                               8.48                                                                              16.81                                                                              24.24 44.67                                                                              30.43 26.17                                                                              20.50 42.60                         480 min                                                                            18.31                                                                               7.61                                                                              14.81                                                                              18.92 39.41                                                                              25.85 22.54                                                                              17.60 37.03                         __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Pharmacokinetic parameters of hydrocortisone after                            subcutaneous administration of HYC.sub.2, HYC.sub.5, HYC.sub.8 at a           dose of 6.5 mg/kg in comparison to the corresponding dose                     of hydrocortisone (1 mg/kg)                                                   PARAMETERS                                                                             HYDROCORTISONE                                                                              HYC.sub.2                                                                             HYC.sub.5                                                                             HYC.sub.8                              __________________________________________________________________________    K.sub.e  0.15 h.sup.-1 0.17 h.sup.-1                                                                         0.24 h.sup.-1                                                                         0.19 h.sup.-1                          t 1/2 elim.                                                                            4.5 h         4.08 h  2.89 h  3.65 h                                 k.sub.a                0.86 h.sup.-1                                                                         0.65 h.sup.-1                                                                         0.94 h.sup.-1                          t.sub.max                                                                              30 min.       2.35 h  2.4 h   2.13 h                                 (AUC).sub.8 .sup.0.5                                                                   192.00 ng/ml h                                                                              241.06 ng/ml h                                                                        278.92 ng/ml h                                                                        250 ng/ml h                            Bioavailability                                                                        70.3          88.3%   100%    91%                                    __________________________________________________________________________

Conclusions

The bioavailability, as compared to hydrocortisone, of the threeproducts in examination, proves to be complete and even superior to thatof the quick release preparation. Regarding this, however, theabsorption is slower (maximum time about 2 hrs) and maximumconcentrations equal to those of subcutaneously administered cortisolare not reached. The plasmatic cortisolemia proves however on average tobe higher several hours after administration. Esterification withhyaluronic acid therefore determines slower release of hydrocortisone,and this is the desired objective.

3) Skin Hydration Studies

Hydrolysis of the ester bond, as has already been said, is a saturationprocess; that is, it tends towards zero grade kinetics. This, for aretard form, is a very desirable factor, since, by definition, acontrolled release preparation is "a preparation which determines therelease of a constant aliquot of active principle in a given time" andthis is the condition reached by zero grade kinetics.

The skin, due to the complex nature of its physiological functions,cannot be considered as exclusively a passive covering organ, but ratheras a dynamic, polyvalent organ. The complete functional capacity of theskin is fundamentally guaranteed by the presence of an intacthydrolipidic covering and this requires a correct humidity content inthe horny layer, which varies a great deal according to its storagecapacity (values vary between 10% and 60% of water content). The skin'shumidity depends on a series of endogenous and exogenous factors.

Cutaneous humidity fundamentally influences the formation of the skin'sspecific hydrolipidic film which modifies and stores the substances iteliminates, thus forming the basis for the realization of its protectivefunctions.

The means of defense used so far to restore the maximum degree ofhydration for the skin involve the use of highly hygroscopic substances,such as glycerine, sodium lactate and propylenic glycol. Thesesubstances, however, had the disadvantage, in dry conditions, of drawinghumidity from the skin itself instead of from the external environment,thus making the skin even drier.

For this reason at present there is a preference for biologicalsubstances whose origins lie, for their particular characteristics, tothe natural hydrating factors mentioned before. In this context isincluded the considerable interest in the use of hyaluronic acid.

The hydration of the skin and its nourishment seem closely related tothe HY content of the cutaneous tissue. It has in fact been demonstratedthat the exogeneous contribution of HY contributes noticeably to thestate of hydration of the cutaneous tissue.

These particular characteristics of hyaluronic acid are also found, andto an even greater degree, in the esterified derivatives of HY accordingto the present invention, and for this reason they may be used to agreat extent in the field of cosmetics.

In order to establish a comparison between hyaluronic acid and itsderivatives of the present invention, some experiments were carried outto instrumentally evaluate, after topical application, the hydratingproperties of the compounds in examination.

Materials

As hyaluronic derivatives according to the present invention thefollowing esters were used.

HYAFF₂ hyaluronic acid esterified by 75% with methanol

HYAFF₇ hyaluronic acid esterified by 75% with ethanol

HYAFF₈ hyaluronic acid esterified by 50% with isopropanol

HYAFF₉ hyaluronic acid esterified by 50% with n-propanol

HYAFF₁₀ hyaluronic acid esterified by 50% with n-butanol

Hyaluronic acid sodium salt (Hyalastine fraction)

All the compounds were vehicled at a concentration of 0.2% in anointment the composition of which was as follows:

Polyethylenglycol monostearate 400, gr. 10.000

Cetiol V, gr. 5.000

Lanette SX, gr. 2.000

Paraoxybenzoate of methyl, gr. 0.075

Paraoxybenzoate of propyl, gr. 0.050

Sodium dehydroacetate, gr. 0.100

Glycerine F.U., gr. 1.000

Sorbitol 70, gr. 1.500

Test cream, gr. 0.050

Water for injectable prepar. q.b.a., gr. 100.000

The placebo formulation contained only vehicle.

Method Study Sample

The study was carried out on 10 healthy volunteers (6 women and 4 mennot suffering from any form of skin disease), aged between 20 and 60years.

Treatment

Each volunteer was treated (single administration) with all theformulations in examination, which were applied (1 gr./ointment) to theinside surface of each forearm, distinguishing with a dermographicpencil the application zone (about 25 cm²) of each product andstandardizing the procedure as far as possible. To the right forearmwere applied the compounds known as HYAFF₂, HYAFF₇, HYAFF₈, HYAFF₉,while to the left were applied HYAFF₁₀, placebo and hyaluronic acid.

Evaluation Parameters

At the established times (0, 3, 6 and 24 hours after treatment) thedegree of hydration was measured with a corneometer of the horny layerof each application zone.

Most particularly, the dielectric strength of the water was measured (in0.8 seconds), after application of the sensor (condenser) to the skinsurface. The value thus obtained, the measurement unit of whichcorresponds to 0.07 mg of water (normal values are between 90 and 100units), was read on the dial of the instrument.

Registrations were carried out in constant humidity conditions.

Results

As can be seen from the results reported in Table 5, treatment with thecompounds of the HYAFF series induced, in all cases, a notable increasein the degree of hydration of the horny layer, which was particularlyevident not only during the hours immediately following application, butalso from the last registrations. This effect proved to be superior bothto that of the placebo formulation and to the formulation containinghyaluronic acid sodium salt. Of the compounds tested, the derivativesHYAFF₂ and HYAFF₉ appeared particularly interesting.

Conclusions

On the basis of the results obtained it was possible to conclude thatthe esterified HYAFF derivatives do in fact determine a notable andprolonged hydrating effect at the skin level, which is superior to thatobserved with the formulation containing hyaluronic acid, thusguaranteeing the integrity and physiological efficiency of thehydrolipidic film. These satisfactory results form therefore a validbasis for the use of these compounds in the prevention (or treatment) ofchapped skin, the treatment of burns and scalds and the maintaining ofphysiological nourishment and elasticity of the skin.

                  TABLE 5                                                         ______________________________________                                        Effect of the compounds of the HYAFF series on the degree                     of hydration of the corneal layer (each value                                 represents the average for 10 subjects)                                                     - x Δ%                                                                           - x Δ%                                                                           - x Δ%                                                                        - x Δ%                            PRODUCT       1st hr   3rd hr   6th hr                                                                              24th hr                                 ______________________________________                                        PLACEBO       41.5     28.3     13.4  2.2                                     HYALURONIC ACID                                                                             54.7     37.6     19.7  5.3                                     HYAFF.sub.2   66.6     43.1     24.5  6.1                                     HYAFF.sub.7   93.5     66.7     30.1  11.4                                    HYAFF.sub.8   70.0     51.4     26.2  8.7                                     HYAFF.sub.9   81.0     59.9     29.0  9.6                                     HYAFF.sub.10  68.8     57.0     27.5  8.3                                     ______________________________________                                    

4) Enzyme Stability and Oxygen Permeability Studies. Materials

The valuable properties of the new esters according to the presentinvention, already partially described, which form their technicaladvantages over the already known products in the respective fields arefurther illustrated by the following results on the stability of theenzymes and the permeability to oxygen of the films obtained with thefollowing compounds:

HYAFF₂ hyaluronic acid esterified by 100% with methanol

HYAFF₇ hyaluronio acid esterified by 100% with ethanol

HYAFF₈ hyaluronic acid esterified by 100% with isopropanol

HYAFF₉ hyaluronic acid esterified by 100% with n-propanol

HYAFF₁₁ hyaluronic acid esterified by 100% with benzylic alcohol

HYAFF₂₀ hyaluronic acid esterified by 100% with β-phenylethylic alcohol

HYAFF₂₂ hyaluronic acid esterified by 100% with isopentylic alcohol

The films may be prepared according to the method described in Example39.

Stability to Enzymes of the HYAFF Films Stability to Serum Esterage

Each film (weighing about 20 mg.) was placed in a polyethylene capsuletogether with 5 ml of rabbit serum and kept at a constant temperature(37° C.).

The evaluation parameter was the time taken (in hours) for the film todissolve. The results are reported in Table 6.

Stability to hyaluronidase

Each film (weighing about 20 mg.) was placed in a polyethylene capsuletogether with pH 5 buffer (acetate 0.1M, NaCl 0.15M) or pH 7.2(phosphate 0.1M, NaCl 0.15M) containing 100 U of enzyme (testiclehyaluronidase from Miles batch 8062, activity 342 turbidometricunits/mg) in each ml and kept at a constant temperature (37° C.). Theevaluation parameter was the time taken (in hours) for the film todissolve. The results are reported in Table 6.

                  TABLE 6                                                         ______________________________________                                        Stability of the films of the HYAFF series                                    derivatives in the presence of serum esterase                                 (37° C.) and in the presence of hyaluronidase                          (37° C.; pH 5 and pH 7.2)                                                                      STABILITY (hrs)                                                               HYALUR-                                               COMPOUNDS               ONIDASE                                               SERUM ESTERASE          pH 5   pH 7.2                                         ______________________________________                                        HYAFF.sub.2    72       120    120                                            HYAFF.sub.9   120       168    168                                            HYAFF.sub.7    90       150    150                                            HYAFF.sub.11   60       140    140                                            HYAFF.sub.20  130       180    180                                            HYAFF.sub.22  130       175    175                                            ______________________________________                                    

Permeability to oxgycen of the films of the HYAFF series

Each film was placed in a container having 2 compartments separated bythe membrane itself. One compartment (volume=1, 2 cc) was filled withpartially degassed water (PO₂ =45 mm of Hg at 23° C.), into the otherwas introduced a flow of O₂ and CO₂ (95% and 5% respectively), keptconstant (1 bubble/second) in time. The whole system was insulated innitrogen.

At the established times (15, 30, 60, 90, 120, 240 minutes) a suitablealiquot of water was drawn off (1.2 cc) and determination of the partialpressure of O₂ was effected by a Gas System analyser 1302 from theInstrumentation Laboratories. The saturation pressure (550 mg of Hg) wastaken as reference value and calculated, in the previously describedexperimental conditions, by insufflating the O₂ atmosphere.

The tests were carried out in comparison to an impermeable membrane andsilastic (Lepetit cat. No. 500-1). The results are reported in Table 7.

                  TABLE 7                                                         ______________________________________                                        PERMEABILITY TO O.sub.2 OF THE FILMS                                          OF THE HYAFF SERIES                                                                   PRESSURE OF O.sub.2 (mm Hg at 23° C.)                          COM-      0      15      30   60   90    120  240                             POUNDS    min    min     min  min  min   min  min                             ______________________________________                                        non perm. 45      90      90   90   90    90   90                             membrane                                                                      Silastic  45     340     406  422  422   422  430                             HYAFF.sub.2                                                                             45     226     285  300  348   390  390                             HYAFF.sub.9                                                                             45     250     303  320  371   427  420                             HYAFF.sub.7                                                                             45     241     298  315  330   340  386                             HYAFF.sub.11                                                                            45     260     310  328  350   400  400                             HYAFF.sub.20                                                                            45     215     256  317  337   380  375                             HYAFF.sub.22                                                                            45     249     288  310  352   373  370                             ______________________________________                                    

PHARMACEUTICAL PREPARATIONS

One object of the present invention is the provision of pharmaceuticalpreparations containing one or more of the above mentioned esters ofhyaluronic acid and salts thereof, or one or more medicaments resultingfrom the association of one of such esters with a pharmacologicallyactive substance for topical application, as described above. That ismedicaments in which the hyaluronic ester acts as a vehicle for theactive substance.

The pharmaceutical preparations containing the hyaluronic esters as anactive principle, both in the case of esters with therapeuticallyinactive alcohols destined for the same uses as hyaluronic acid itself,and esters with therapeutically active alcohols intended for the usualuses of such alcohols, contain the usual excipients and may be employedfor oral, rectal, parenteral, subcutaneous, local or intradermal use.They are therefore in solid or semisolid form, for example as pastilles,tablets, gelatin capsules, capsules, suppositories, soft gelatincapsules. For parenteral and subcutaneous use it is possible to useforms intended for intramuscular or intradermal administration, orsuitable for infusions or intravenous injections. It is possibletherefore to provide solutions of the active compounds or freeze driedpowders of the active compounds to be added to one or morepharmaceutically acceptable excipients or diluents, convenient for theabove mentioned uses and with osmolarity compatible with thephysiological liquids. For local use, preparations in spray form may beused, for example nasal sprays, creams or ointments for topical use orspecially prepared sticking plasters for intradermal administration. Thesolubility of the hyaluronic esters in organic solvents with low boilingpoints makes them particularly suitable for the manufacture of "sprays".

The preparations of the invention may be administered to man or animals.They contain preferably between 0.01% and 10% of active component forthe solutions, sprays, ointments and creams and between 1% and 100%,preferably between 5% and 50%, of active compound for the preparationsin solid form. The dosage to be administered will depend on individualneeds, on the desired effect and on the chosen administration route. Thedaily dosage of such preparations may be decided according to that usefor the corresponding known preparations both of hyaluronic acid for thecorresponding cures, for example for the cure of arthritis, for examplein man or horse, and of the therapeutically active alcohol, the actionof which is to be put to use. Thus, for example, the dosage of ahyaluronic ester with cortisone may be derived from its content of thissteroid and from its usual dosage in the known pharmaceuticalpreparations.

One particular form of pharmaceutical preparations is represented by theabove mentioned medicaments containing the association of an hyaluronicester and of one or more active substances. These may also be in solidform, for example as freeze dried powders containing only the twocomponents (1) and (12), together or separate. This galenic form isespecially suitable for topical use. Indeed these solid medicamentsform, on contact with the surfaces to be treated, more of lessconcentrated solutions according to the nature of the particularepithelium, with the same characteristics of the solutions previouslyprepared in vitro and which represent another particularly importantaspect of the present invention. Such solutions are preferably indistilled water or sterile saline and preferably contain no otherpharmaceutical vehicle apart from the hyaluronic ester or one of itssalts. The concentrations of such solutions may also vary within amplelimits, for example between 0.01 and 75% both for each of the twocomponents taken separately, and for their mixtures or salts. Particularpreference is given to solutions with a pronounced elastic-viscouscharacter, for example with a content of 10% to 90% of the medicament orof each of its components.

Particularly important are the medicaments of this type, both inanhydrous form (freeze dried powder) or as solutions, concentrated ordiluted in water or saline, possibly with the addition of additive orauxillary substances, such as in particular disinfectant substances ormineral salts acting as buffer or others, for ophthalmic use.

Of the medicaments of the invention, particularly important, as the casemay be, are those with a degree of acidity suitable for the environmentto which they are to be applied, that is with a physiologicallytolerable pH. Adjustment of the pH, for example in the above mentionedsalts of the esters of hyaluronic acid with a basic active substance,may be effected by suitably regulating the quantities of polysaccharide,of the salts of the basic substance itself. Thus, for example, if theacidity of a salt of a hyaluronic ester with a basic substance is toohigh, the excess of free acid groups is neutralized with the abovementioned inorganic bases, for example with sodium, potassium orammonium hydrate.

The preparation of the salts according to the invention may be effectedin a per se known manner by placing in contact solutions, aqueoussolutions or organic solutions, of the two components (1) and (2), andpossibly bases or basic salts of the above mentioned alkaline oralkaline earth metals or magnesium or aluminum in the right quantitiesand isolating the salts in an amorphous anhydrous form according toknown techniques. It is possible for example to prepare first aqueoussolutions of the two components (1) and (2), freeing such componentsfrom aqueous solutions of their salts with suitable ionic exchangers,and mixing the two solutions at a low temperature, for example between0° C. and 20° C. If the salt thus obtained is easily soluble in water itcan be freeze dried, while the salts which are difficult to solubilizemay be separated by centrifugation, filtration or decantation andpossibly subsequently dried.

For these associated medicaments too, the dose is based on that of theactive principles used singly and may therefore easily be determined bya skilled person, taking into consideration the dosages recommended forthe corresponding known drugs.

In the cosmetic articles according to the invention the hyaluronicesters and their salts are mixed with the excipients commonly used inthe field and are for example those already listed above for thepharmaceutical preparations. Mostly used are creams, ointments, andlotions for topical use in which the hyaluronic ester or one of itssalts may represent the cosmetic active principle possibly with theaddition of other cosmetically active principles, such as for examplesteroids, for example pregnenolone, or one of the principles mentionedabove. In such preparations the hyaluronic ester is preferably an esterwith an alcohol with no cosmetic action, such as an lower aliphaticalcohol, such as one of those already mentioned. The effect is due tothe intrinsic cosmetic properties of the polysaccharide component, suchas in the case of free hyaluronic acid or of its salts.

The cosmetic articles may however be based on substances with specificactions other than those of hyaluronic acid, for example disinfectantsubstances, sunshields, waterproofing or regenerating or antiwrinklesubstances, or odorants, especially perfumes. In this case thehyaluronic ester may itself be the active ingredient and derives fromalcohols with the same properties, for example from higher aliphaticalcohols or terpenic alcohols in the case of perfumes or acts above allas vehicle for substances with those properties associated with it.

Particularly important therefore are cosmetic compositions similar tothe medicaments described above in which the pharmaceutically activecomponent (1) is substituted by a cosmetic factor, and the relativesalts.

The use of the above mentioned esters deriving from alcohols used in theperfume industry represent an important step forward in technology,since they allow a slow, constant and protracted release of the scentedingredients.

An important application of the present invention regards sanitary andsurgical articles already described above, the methods for theirmanufacture and use. The invention therefore includes all the articlessimilar to those already on the market, based on hyaluronic acid butcontaining a hyaluronic ester or one of its salts in place of the freeacid or one of its salts, for example inserts or ophthalmic lenses.

Completely new surgical and sanitary articles according to the presentinvention are represented by the esters of hyaluronic acid regeneratedas such by appropriate organic solutions from which it is possible toobtain, by means of the suitable procedures, films, thin sheets orthreads to be used in surgery, as aids or substitutes of the skin incases of serious damage to this organ, such as for example followingburns, as a suture in surgical operations. The invention includesparticularly these uses and a procedure for the preparation of sucharticles consisting in the formation of a solution of hyaluronic esteror of one of its salts in a suitable organic solvent such as an amide ofa carboxylic acid, especially a dialkylamide of an aliphatic acid withbetween 1 and 5 carbon atoms and deriving from alkyl groups with between1 and 6 carbon atoms, and above all from an organic sulphoxide, that isa dialkylsulphoxide with alkyl groups with a maximum of 6 carbon atoms,such as especially dimethylsulphoxide or diethylsuphoxide and again mostimportantly by a fluoronated solvent with a lower boiling point such asespecially hexafluoro-isopropanol. The invention includes turning suchsolutions into sheet or thread form and in removing the organic solventby contact with another organic or aqueous solvent which can be mixedwith the first solvent and in which the hyaluronic ester is not soluble,especially a lower aliphatic alcohol, for example ethyl alcohol (wetspinning), or, should a solvent with a not too high boiling point beused to prepare the solution of the hyaluronic derivative, in removingsuch a solvent in dry conditions with a current of gas, especially withsuitably heated nitrogen (dry spinning). It is also possible to obtainexcellent results with dry-wet spinning.

The threads obtained with hyaluronic acid esters may be used for thepreparation of gauzes for the medication of wounds and in surgery. Theuse of such gauzes has the except ional advantage of theirbiodegradability in the organism, made possible by the enzymes whichthey contain. These enzymes divide the ester into hyaluronic acid andthe corresponding alcohol, and therefore into a compound already presentin the organism, made possible by the enzymes which they contain. Theseenzymes divide the ester into hyaluronic acid and the correspondingalcohol, and therefore into a compound already present in the organismand into a harmless compound, such as an alcohol, should a hyaluronicester be used which derives from a therapeutically acceptable alcohol,such as ethyl alcohol.

These gauzes and also the aforementioned threads may therefore be leftinside the organism after surgery, since they are slowly absorbed thanksto the aforesaid degradation.

During the preparation of the sanitary and surgical articles describedabove, it is possible to add plastifying materials which improve theirmechanical characteristics, such as in the case of the threads, toimprove their resistance to knots. These plastifying materials may befor example alkaline salts of fatty acids, for example sodium stearateor sodium palmitate, the esters of organic acids with many carbon atoms,etc.

Another application of the new esters, using to advantage theirbiodegradability due to the esterases present in the organism, isrepresented by the preparation of capsules for subcutaneous implantationof medicaments or of microcapsules for injection, for example bysubcutaneous or intramuscular route. For the applications ofsubcutaneous medicaments for obtaining a slow release and therefore a"retard" action, capsules made of silicone materials have mostly beenused up till now, with the disadvantage that the capsule is liable tomove about inside the organism and it is not possible to recover it.Evidently with the new hyaluronic esters this danger no longer exists.

Of great importance is also the preparation of microcapsules made withhyaluronic esters, eliminating the problems regarding their use which uptill now has been limited, for the same reasons as those mentioned aboveand which opens up a vast field of application where a "retard" effectis sought be an injected route.

A further application in the sector of medicine and surgery of the newesters concerns the preparation of a large variety of solid inserts suchas plates, discs, sheets, etc. substituting those in metallic form orthose made of synthetic plastic materials already in use, in the case ofinserts intended for removal after a certain period of time.Preparations made of animal pollagen, being of a proteic nature, oftenprovoke undesirable side effects such as inflammation or rejection. Inthe case of animal, and not human, hyaluronic acid, this danger does notexist, as there is no incompatability between the polysaccharides ofdifferent animal species.

Another application relates to the use to augment and correct softtissue defects. The need for a safe and effective biomaterial by whichto replace missing or damaged soft tissue has long been recognized.Several alloplastic materials, including paraffin, Teflon paste,silicone and borine collagen have been used to replace lost soft tissue.However, these materials have been associated with permanent undesirabletextural changes in the skin, with migration from the site ofimplantation and with adverse treatment reactions. Thus, the need for aversatile biomaterial in medicine continues. The hyaluronic acid esterscan be used safely and effectively to augment and correct such softtissue defects as acne scars, atrophy post surgical irregularities, mohschemiosurgery, cleft lip sears and age-related wrinkles.

Part of the applications in the field of medicine and surgery of the newesters according to the present invention, are represented by expansivematerials, especially in the form of sponges, for the medication ofwounds and various lesions.

The following are particular exemplary pharmaceutical preparationsaccording to the invention.

Formulation 1--Collirium containing cortisone of which 100 ml contain:

partial ester of hyaluronic acid with cortisone (Ex. 10), gr. 0.200

ethyl p. hydroxybenzoate, gr. 0.010

methyl p. hydroxybenzoate, gr. 0.050

sodium chloride, gr. 0.900

water for injectable preparations/q.b.a., ml. 100

Formulation 2--Injectable solution containing hydrocortisone of which100 ml contain:

partial ester of hyaluronic acid with hydrocortisone (Ex. 11), gr. 0.1

sodium chloride, gr. 0.9

water for injectable preparations/q.b.a., ml. 100

Formulation 3--Cream containing a partial ester of hyaluronic acid withethyl alcohol (Ex. 3), of which 100 gr. contain:

partial ester of hyaluronic acid with ethyl alcohol, gr. 0.2

Polyethylenglycol monostearate 400, gr. 10.000

Cetiol V, gr. 5.000

Lanette SX, gr. 2.000

Paraoxybenzoate of methyl, gr. 0.075

Paraoxybenzoate of propyl, gr. 0.050

Sodium dihydroacetate, gr. 0.100

Glycerine F.U., gr. 1.500

Sorbitol 70, gr. 1.500

Test cream, gr. 0.050

Water for injectable preparations/q.b.a., gr. 100.00

The following are exemplary material products utilizing the hyaluronicesters of the invention.

EXAMPLE 39 Preparation of films using esters of hyaluronic acid

A solution is prepared in dimethylsulfoxide of the n-propyl ester of HY(MW 130,000) with a concentration of 180 mg/ml.

By means of a stratifier, a thin layer of solution is spread on a glasssheet; the thickness must be 10 times greater than the final thicknessof the film. The glass sheet is immersed in ethanol which absorbs thedimethylsulfoxide but does not solubilize the HY ester which becomessolid. The film is detached from the glass sheet, is repeatedly washedwith ethanol, then with water and then again with ethanol.

The resulting sheet is dried in a press for 48 hours at 30°.

EXAMPLE 40 Preparation of threads using esters of hyaluronic acid

A solution is prepared in dimethylsulfoxide of the benzyl ester of HY(MW 165,000) with a concentration of 200 mg/ml. The solution thusobtained is pressed by means of a pump through a threader with 0.5 mmholes.

The threader is immersed in ethanol/dimethylsulfoxide 80:20 (thisconcentration is kept constant by continuous addition of ethanol); whenthe solution in dimethylsulfoxide is soaked in this way it tends to losemost of the dimethylsulfoxide and the thread solidifies.

The thread is stretched while it still has a content ofdimethylsulfoxide, is then repeatedly stretched and washed with ethanol.The thread is dried in nitrogen current.

EXAMPLE 41 Preparation of a spongy material made with hyaluronic acidesters

1 g of benzyl ester of hyaluronic acid with a molecular weight of170,000 in which all the carboxylic groups are esterified (obtained forexample as described in Example 14) are dissolved in 5 ml ofdimethylsulfoxide. To each 10 ml of solution prepared, a mixture of 31.5g of sodium chloride with a degree of granularity corresponding to 300μ,1.28 g of sodium bicarbonate and 1 g of citric acid is added and thewhole is homogenized in a mixer.

The pasty mixture is stratified in various ways, for instance by meansof a mange consisting of two rollers which turn opposite each other atan adjustable distance between the two. Regulating this distance thepaste is passed between the rollers together with a strip of siliconepaper which acts as a support to the layer of paste thus formed. Thelayer is cut to the desired dimensions of length and breadth, removedfrom the silicone, wrapped in filter paper and emerged in a suitablesolvent, such as water. The sponges thus obtained, are washed with asuitable solvent such as water and possibly sterilized with gamma rays.

EXAMPLE 42 Preparation of a spongyv material made with hyaluronic acidesters

In the manner described in Example 41, it is possible to prepare spongymaterials with other hyaluronic acid esters. In the place ofdimethylsulfoxide it is possible to use, if desired, any other solventcapable of dissolving the chosen ester. In the place of sodium chlorideit is possible to use any other solid compound which is insoluble in thesolvent used to dissolve the hyaluronic acid ester, but which is howeversoluble in the solvent used to precipitate the hyaluronic ester afterthe above mentioned mechanical treatment, and finally which has thecorrect degree of granularity to obtain the type of pores desired in thesponge material.

In the place of sodium bicarbonate and citric acid it is possible to useother couples of similar compounds, that is, compound which react toeach other in suspension or solution of the solvent used to dissolvehyaluronic acid in such a way as to form a gas, such as carbon dioxide,which has the effect of producing a less compact spongy material. Inthis way it is possible to use, in the place of sodium bicarbonate,other bicarbonates or alkaline or alkaline earth carbonates and in theplace of citric acid other acids in solid form, such as tartaric acid.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

We claim:
 1. A partial ester of hyaluronic acid with an alcohol of thealiphatic, araliphatic, cycloaliphatic or heterocyclic series wherein atleast a first portion of the carboxylic acid groups of said hyaluronicacid are salified with a therapeutically active amine.
 2. A partialester of hyaluronic acid according to claim 1, wherein a second portionof the carboxylic acid groups of said hyaluronic acid is salified withan alkaline or alkaline earth metal, magnesium or aluminum.
 3. A partialester of hyaluronic acid according to claim 1, wherein saidtherapeutically active amine is a member selected from the groupconsisting of alkaloids, peptides, phenothiazines, benzodiazepines,thioxanthenes, hormones, vitamins, anticonvulsants, antipsychotics,antiemetics, anesthetics, hypnotics, anorexics, tranquilizers, musclerelaxants, coronary vasodilators, antineoplastics, antibiotics,antibacterials, antivirals, antimaralarials, carbonci anhydraseinhibitors, nonsteroid antiinflammatory agents, vasoconstrictors,cholinergic agonists, cholinergic antagonists, adrenergic agonists,adrenergic antagonists, and narcotic antagonists.
 4. A partial esteraccording to claim 1, which is in the form of a microcapsule.
 5. Apartial ester according to claim 3, which is in the form of amicrocapsule.